CN104056261B - Indoleamine 2, 3-dioxygenase based immunotherapy - Google Patents

Indoleamine 2, 3-dioxygenase based immunotherapy Download PDF

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CN104056261B
CN104056261B CN201410247727.XA CN201410247727A CN104056261B CN 104056261 B CN104056261 B CN 104056261B CN 201410247727 A CN201410247727 A CN 201410247727A CN 104056261 B CN104056261 B CN 104056261B
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ido
cell
hla
peptide
cancer
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CN104056261A (en
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梅斯·哈尔德·安德森
珀·托尔·斯特拉藤
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IO Biotech ApS
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Hvidovre Hospital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
    • C12Y113/11052—Indoleamine 2,3-dioxygenase (1.13.11.52), i.e. indoleamine 2,3-dioxygenase 1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response

Abstract

The present invention relates to the field of prophylaxis and therapy of cancer. In particular there is provided a protein lndoleamine 2,3-dioxygenase (IDO) or peptide fragments here of that are capable of eliciting anti-cancer immune responses. Specifically, the invention relates to the use of IDO or peptides derived here from or IDO specific T-cells for treatment of cancer. The invention thus relates to an anti-cancer vaccine which optionally may be used in combination with other immunotherapies and to IDO specific T-cells adoptively transferred or induced in vivo by vaccination as a treatment of cancer. It is an aspect of the invention that the medicaments herein provided may be used in combination with cancer chemotherapy treatment. A further aspect relates to the prophylaxis and therapy of infections by the same means as described above. The use of IDO and immunogenic peptide fragments hereof in cancer and infection treatment, diagnosis and prognosis is also provided.

Description

Immunotherapy based on indole amine 2,3-dioxygenase
The application is filing date on April 17th, 2009, application artificial " Hai Laiwu hospital ", invention entitled " based on Yin Diindyl amine 2, the immunotherapy of 3-dioxygenase " the divisional application of Chinese patent application 200980122844.0.
Cross-Reference to Related Applications
The all patents applied at this or quote in the application and the full content of non-patent reference are also by quoting It is herein incorporated.
Technical field
The present invention relates to the prevention of cancer and the field for the treatment of.Especially, it is provided that antitumor immune response can be caused Protein IDO (IDO) or its fragments of peptides.In particular it relates to IDO or spread out by it Raw peptide or the application in treatment cancer of the IDO specific T-cells.Therefore the present invention relates to anti-cancer vaccine, it is the most permissible Be used in combination with other immunotherapies, and relate to adoptive transfer or the IDO specificity T induced by vaccination in vivo Cell is as cancer treatment method.One aspect of the present invention is that medicine provided herein can be combined make with cancer chemotherapy treatment With.Further aspect relate to by same way as above to infect prevention and treatment.
Additionally provide IDO and the application in cancer and treatment of infection, diagnosis and prognosis of its immunogenic peptide portions.
Background technology
Immune system has the ability identifying and destroying neoplastic cell；But, although neoplastic transformation and immunogenicity The fact that expression of antigen is correlated with, but immune system often can not be effectively to these antigen responses.Immune system is the most resistance to By these antigen1.When this happens, neoplastic cell breeds uncontrollably, causes the formation of malignant cancer, wherein Its prognosis of individuality for getting involved is very poor.Success for cancer immunotherapy is carried out, it is necessary to overcome acquired tolerance status.
IDO (IDO) is the main component maintaining immune homeostasis, but it also promotes The toleration of tumor inducing.The expression of IDO and activating in tumor and tumor-draining lymphode (LN) via essential amino acids color The degraded of propylhomoserin directly suppresses T cell or the immunosuppressant enhancing mediated via local T reg-(activating regulatory T-cells) Set up tolerogenic environment.For the former, some biological actions of IDO exhaust mediation by the local of tryptophan, and Other effects then mediate through immune regulative tryptophan metabolism thing3,4.In recent years, having identified the IDO-in T cell should Answering signal transducting system, it includes stress kinases GCN2 5.The GCN2 increase generation response to neutral tRNA, if T is thin Born of the same parents exhaust tryptophan, and this will occur, and lacks suppression and the anergy induction of the T cell tolerance IDO mediation of GCN26。
IDO can be expressed by tumor cell and stromal cells in intra-tumor, suppresses the effect of immunne response this its Ying Qi.It addition, the antigen-presenting cell (APC) expressing IDO is present in tumor-draining lymphode, think that at this they establish Tolerogenic microenvironment.It practice, the CD19 of the expression IDO separated from tumor-draining lymphode+Plasmacytoid dendritic cells (DC) immunosuppressant and the T cell anergy of complexity are mediated in vivo7,8；From Normal Lymph Nodes and the Plasmacytoid of spleen DC does not express IDO.Express IDO to cellularity considerably less in the normal lymphoid tissue in addition to intestinal.This implies tumor DC in draining lymph node, constitutive expression IDO must receive stimulation, and this stimulation is relevant with the existence of tumor.Think This stimulation is to be transmitted by from the activating regulatory T-cells (Tregs) of tumor migration to draining lymph node.Tregs has shown that IDO can be induced via the cell surface expression of CTLA-49.The induction of IDO by tumor-draining lymphode from immunity environment transition Become tenable environment.It practice, work as IDO+When DC is injected to internal, it is special that they set up antigen in the lymph node of drain injection site The suppression of specific T cell and anergy10.In addition to its expression in immunologically competent cell, IDO is usually in tumor micro-loop Border is expressed, or in tumor cell itself, or in different stromal cells.In the case, IDO is considered suppression immunity The effector phase of response11,12.In clinic, observing the most repeatedly, in tumor cell, the expression of IDO has with the best prognosis Close13,14。
Therefore, the expression of IDO and the cancer immunity suppression of adjoint IDO induction cause the problem in treatment of cancer.
Summary of the invention
The present invention is based on inventor has now surprisingly been found that the problem solving cancer immunity suppression, and this is the discovery that in cancer For the spontaneous cytotoxicity immunne response of IDO express cell in disease patient.These discoveries open novel therapeutic and diagnosis The road of mode, it can be widely applied in the control of Cancerous disease.
The present invention is by directly killing the cancerous cell expressing IDO and expressing IDO, the APC/ of induction anergy by killing DC carrys out target on cancer disease.This cell expressing IDO by making T cell be capable of identify that completes.Similarly, when clinical disease it is During infection, T cell can kill the APC/DC expressing IDO.Therefore, in cancerous cell and APC the expression of immunosuppressant enzyme IDO and this The application of the method for invention combines positive effect, the cell of the method targeting of the present invention these expression IDO.The method, Especially because it must kill APC/DC, contrary with the prevailing paradigm in this area, in the prevailing paradigm of this area generally Attempting to lower/suppress IDO so that the tenable environment removed around APC/DC retains these cells simultaneously, this is considered as necessary To cause effective immunne response.
Additionally, for express IDO cell spontaneous cytotoxicity immunne response be the discovery that particularly surprising, Because expressing the required effect of cell other immunotherapy methods of antagonism of IDO.Therefore, targeting IDO and the immunity of target tumor The combination of therapy is high Collaboration.
The present invention relates to a kind of vaccine combination as medicine, it comprises SEQ ID NO:(1,13,14,15 and/or 16) IDO (IDO) or with SEQ ID NO:(1,13,14,15 and/or 16) to have at least 70% same Its function congener of property or comprise the immunogenic activity fragments of peptides of continuous sequence of its function congener of described IDO or described Or encode the nucleic acid of described IDO or described fragments of peptides；And adjuvant.
One aspect of the present invention provides the cooperative effect of the combination of immunotherapy based on vaccine disclosed above, should Aspect relates to comprising this vaccine combination and the multicomponent kit of other immunostimulatory compositions.
The aspect that the vaccine of the present invention with other treatment of cancer such as chemotherapeutics be combined is also provided herein.
It is also provided herein the vaccine of the present invention and other treatment such as immunotherapy and/or antibiosis for infection The aspect that element combines.
Another aspect of the present invention relate to a kind of in vitro or in-situ diagnostics cancer patient at PBL or at tumor tissues In comprise SEQ ID NO:(1 with the compositions of the existence of the T cell of responding property of IDO, described compositions, 13,14,15 and/or 16) IDO (IDO) or with SEQ ID NO:(1,13,14,15 and/or 16) to have at least 70% same Its function congener of property or comprise the immunogenic activity peptide sheet of continuous sequence of its function congener of described IDO or described Section.
An aspect additionally relate to a kind of in vitro or in-situ diagnostics cancer patient at PBL or in tumor tissues The diagnostic kit of the existence with the T cell of responding property of IDO, described test kit comprises SEQ ID NO:(1,13,14,15 and/ Or 16) IDO (IDO) or with SEQ ID NO:(1,13,14,15 and/or 16) have at least 70% Its function congener of homogeneity or comprise the immunogenic activity peptide of continuous sequence of its function congener of described IDO or described Fragment.
Fragments of peptides and the I class of a kind of continuous sequence that comprise described IDO or described its function congener are also provided herein The complex of the fragment of HLA or II class HLA molecule or this quasi-molecule.
Another aspect of the present invention is a kind of to detect the method for the existence of IDO reaction-ive T cell in cancer patient, described Method includes the fragments of peptides of tumor tissues or blood sample and the continuous sequence comprising its function congener of described IDO or described and I The complex contacts of the fragment of class HLA or II class HLA molecule or this quasi-molecule, and detect described complex and described tissue or The combination of described hemocyte.
A further aspect of the invention relate to a kind of can be with the continuous sequence comprising its function congener of described IDO or described The specific binding molecule of fragments of peptides of row.
As can be seen here, treat the method for clinical disease such as cancer or infection by any one above-mentioned mode and be included in this In the range of invention；Described mode includes the vaccine as above combination using effective dose to the individuality suffering from clinical disease Thing；Molecule that can be specific binding with the fragments of peptides of the continuous sequence comprising its function congener of described IDO or described, or bag Containing above-mentioned vaccine or molecule and other immunostimulatory compositions and/or the multicomponent kit of chemotherapeutics.
Therefore the immunogenic activity fragments of peptides or above-mentioned of the continuous sequence of its function congener of described IDO or described is comprised Vaccine combination is also the purpose of the present invention in the application manufactured in the medicine for the treatment of or prophylaxis of cancer disease.
Another object of the present invention is a kind of method monitoring immunization, said method comprising the steps of: There is provided from individual blood sample；There is provided SEQ ID NO:(1,13,14,15 and/or 16) IDO or with SEQ ID NO:(1,13, 14,15 and/or 16) there is its function congener of at least 70% homogeneity or comprise its function congener of described IDO or described The immunogenic activity fragments of peptides of continuous sequence or encode the nucleic acid of described IDO or described fragments of peptides；Determine that described blood sample is No comprise the antibody being combined with described protein or peptide specific or containing the T cell that is combined with described protein or peptide specific The T cell of receptor；And so that it is determined that in described individuality, created the immunity for described protein or peptide answer Answer.
Accompanying drawing explanation
Fig. 1 is for the restricted t cell response of HLA-A2-of IDO；
The combination of Fig. 2 a HLA-A2-restricted positive control peptide；
Fig. 2 b is from the IDO5-specific C D8T cell in the PBL of renal cell carcinoma patients；
The tetramer analysis of Fig. 2 c-f IDO5-specific T-cells；
The specificity of Fig. 3 IDO5-specific T-cell clones and functional capabilities；
Fig. 4 rectangular histogram shows that intracellular IDO dyes；
Fig. 5 IDO5-specific T-cell clones (RBS35) kills the functional performance of the breast cancer cell line that IFN-γ processes Power；
Fig. 6 IDO5-specific T-cell clones (RBS35) kills the functional capabilities of DC；
Fig. 7 passes through51Cr-release algoscopy measures specificity and the functional capabilities of IDO5-specific T-cells；
The Multiple Sequence Alignment of Fig. 8 IDO serial ID O, IDOA, IDOB and IDOC；
The pairing comparison of Fig. 9 IDO and IDOLIKE.
Detailed description of the invention
The main object of the present invention is to provide a kind of risk as prophylaxis of cancer, reducing formation cancer or treats cancer The vaccine combination of medicine, it comprises IDO (IDO) or its immuno active polypeptide fragment.
Definition
Adjuvant: the mixing with the immunogenic determinant/antigen/nucleic acid construct used increases or changes in other manners Any material to the immunne response of described determinant.
Aminoacid: any synthesis or naturally occurring amino carboxylic acid, including any aminoacid being present in peptide and polypeptide, Described peptide and polypeptide include protein and the enzyme of internal synthesis, therefore include amino acid whose modification.Term ammonia used herein Base acid with term " amino acid residue " synonym, the implication of amino acid residue comprise the most with at least one other thing Class, such as 2 kinds, such as 3 kinds, the such as aminoacid of other species reaction more than 3 kinds.Generic term aminoacid includes natural With non-natural aminoacid, they any one may be at " D " or " L " isomeric form.
Antibody: immunoglobulin molecules and the active part of immunoglobulin molecules.The immune ball that antibody is the most complete Protein molecular or the fragment of its reservation immunologic competence.
Antigen: any material that can combine with the immunity receptor of Clonal distribution (T cell or B-cell receptor).Generally, For peptide, polypeptide or multimer polypeptide.Antigen is preferably able to cause immunne response.
APC: antigen-presenting cell.APC is the cell showing the exotic antigen compound with MHC in its surface.T cell can To use its φt cell receptor (TCR) to identify this complex.APC is divided into two classes: Professional, (it has a three types: dendritic cell, Macrophage and B cell) or the non-full-time (major histocompatibility needed for constitutive expression does not interacts with Naive T cells Property complex proteins matter；These are only expressed when stimulating non-full-time APC by some cytokine such as IFN-γ).
Strengthen: carried out the immunoreagent strengthening being to give extra dose by booster shot or dosage, such as vaccine, this is exempted from Epidemic disease reagent certain time after predose gives thus keeps the immunne response caused by the identical reagent place of preceding dose.
Cancer: before the most any tumor or neoplastic disease, optimum or pernicious, wherein " tumprigenicity " is phalangeal cell Abnormality proliferation.
Carrier: be combined the entity with helper-inducer immunne response or compound with antigen.
Chimeric protein: the protein of a kind of genetic modification, its by by by two or more completely or part gene Or a series of (non-) random nucleic acid be stitched together preparation nucleotide sequence coded.
Clinical disease: need the disease of medical rescue, especially the most relevant with the expression of IDO disease.Described disease The example of disease includes: cancer and infection.
Complement: the blood protein of series of complex, the effect of its effect " supplementing " antibody.Complement destruction antibacterial, produces inflammation Disease, and regulate immunoreation.
CTL: cytotoxic T lymphocyte.One expresses CD8 and φt cell receptor and therefore, it is possible to by I quasi-molecule The T cell subgroup of the antigen response presented.
Cytokine: growth or differentiation, it uses the most in non-limiting manner, and should be not intended to this Invention and the explanation of claim.In addition to cytokine, adhesion molecule or accessory molecule, or its any combination, Ke Yidan Solely use or be used in combination with cytokine.
Delivery vector: a kind of entity, by this entity, nucleotide sequence or polypeptide or both can be from least one medium It is transported to another kind of medium.
DC: dendritic cell.(DC) it is immunocyte and the part forming immune system.They main Function is process antigen material and is presented on the surface of other cells immune, therefore as antigen-presenting cell (APC) work.
Fragment: be used for representing the non-total length part of nucleic acid or polypeptide.Therefore, fragment itself is nucleic acid or polypeptide the most respectively.
Function congener: function congener can be and the wild type shape of given gene/gene outcome/protein/polypeptide Formula/sequence display at least some sequence iden and retain the functional any nucleic acid in terms of at least one of initiation sequence/ Protein/polypeptide.The function congener of IDO has the ability inducing the immunne response to the cell expressing IDO herein.
IDO: IDO.At SEQ ID NOs:(1,13,14,15, and 16) in identified.
Individual: usually bird, mammal, fish, Amphibian or any kind of reptile or subspecies, preferably suckling Animal, the most preferably mankind.
Infect: term " infects " any kind of clinical disease relating to producing immunne response herein, and therefore includes Infection, chronic infection, autoimmune disease and allergic inflammation.
Separate: " separation " of using with nucleic acid disclosed herein, polypeptide and antibodies refers to these nucleic acid, polypeptide The most identified with antibody and separate and/or reclaim from the component of its natural surroundings (usually cellular environment).The present invention's Nucleic acid, polypeptide and antibody preferably separate, and the vaccine of the present invention and other compositionss preferably comprise the nucleic acid of separation, many Peptide or the antibody of separation.
, there is the MHC of two kinds of major subtypes, I class and II class in MHC: MHC.
Nucleic acid: the chain of the nucleotide conveyed hereditary information or sequence.For the present invention, nucleic acid is DNA (deoxyribonucleic acid) (DNA)。
Nucleic acid construct: the nucleic acid of genetic modification.Typically comprise several element such as gene or its fragment, promoter, Enhancer, terminator, polyA tail, joint, polylinker, operation joint, multiple clone site (MCS), mark, STOP codon, Other controlling elements, internal ribosome entry site (IRES) etc..
Operation joint: by two parts nucleic acid construct or (being fitted together to) in the way of guaranteeing processing biology of nucleic acid or polypeptide Nucleotide that polypeptide combines or the sequence of amino acid residue.
Pathogen: the specific pathogenetic factor of disease, especially biological agent such as virus, antibacterial, Protein virus or parasite, They can make its host cause disease, also referred to as infectious agent (infectious agent).
PBL: peripheral blood cells is the cellular component of blood, and it is made up of erythrocyte, leukocyte and platelet, and it is at blood Circulatory pool in find and be not isolated in lymphsystem, spleen, liver or bone marrow.
PBMC: peripheral blood lymphocytes (PBMC) is the hemocyte with round cell core, such as lymphocyte or monokaryon Cell.These hemocytees are immune systems to infection the key component that adapts to invader.Lymphocyte populations is by T cell (CD4 Positive with CD8～75%), B cell and NK cell (be combined～25%) composition.
Peptide: formation sequence the multiple covalently bound amino acid residue connected by amido link.This term and oligopeptide and many Peptide is similarly used.Natural and/or alpha-non-natural amino acid can connect by peptide bond or by non-peptide bond.Term peptide also includes leading to Cross the post translational modification that chemical or enzymatic reaction introduces, as known in the art.This term can refer to the change of polypeptide Body or fragment.
Pharmaceutical carrier: also referred to as excipient or stabilizer, under the dosage used with concentration its to contact its cell or Individuality is avirulent.Generally, physiology's acceptable carrier is pH aqueous buffer solution.The example of physiology's acceptable carrier Including buffer such as phosphate, citrate and other organic acid；Antioxidant, including ascorbic acid；Low-molecular-weight (is less than About 10 residues) polypeptide；Protein, such as serum albumin, gelatin or immunoglobulin；Hydrophilic polymer such as polyethylene pyrrole Pyrrolidone；Aminoacid such as glycine, glutamine, agedoite, arginine or lysine；Monosaccharide, disaccharide and other carbon water Compound, including glucose, mannose or dextrin；Chelating agen such as EDTA；Sugar alcohol such as mannitol or sorbitol；Form salt Counter ion counterionsl gegenions such as sodium；And/or nonionic surfactant such as TWEEN.TM., Polyethylene Glycol (PEG) and PLURONICS.TM。
Multiple (kinds): at least two (is planted).
Promoter: the binding site in DNA, it is one or more adjacent to start that RNA polymerase is combined in this site Messenger RNA is transcribed by structural gene.
Signal peptide: amino acid whose short sequence, it determines protein final position in cell, also referred to as sorts peptide (sorting peptide)。
Surfactant: the capillary surface-active agents of the liquid dissolving it can be reduced.Surfactant is Containing hydrophilic polar group and the compound of non-polar group that is hydrophobic and that be often made up of aliphatic chain.
Treg: regulatory T cells/T lymphocyte.
Vaccine: a kind of material or compositions that can induce immunne response in animal.Referred to herein as immunogen Property compositions.Immunne response is the immunne response (body fluid/antibody and/or cellular immunization) inducing memory in organism, causes Infectious agent is resisted by secondary response rather than primary response, thus reduces its impact on host organisms.The vaccine of the present invention can To give as preventative and/or curative drug.Said composition can comprise following one or more: one or more resist Former, comprise the nucleic acid construct of one or more antigens being operably connected with Ii, carrier, adjuvant and pharmaceutical carrier.
Variant: " variant " with reference to nucleic acid or polypeptide specified refers to display and described reference nucleic acid or polypeptide certain Sequence homology/the homogeneity of degree but be different from described with reference to nucleic acid or the nucleic acid of polypeptide or polypeptide.
IDO
IDO (IDO) is the enzyme that catalysis L-Trp is changed into N-formylkynurenine, and because of This is first enzyme and the rate-limiting enzyme of tryptophan catabolism by kynurenine pathway.The catabolism of tryptophan causes color The consumption of propylhomoserin, it consumes suppression t cell response and promotes mammal gestation, tumor opposing (tumor Resistance) immunologic tolerance in, chronic infection, autoimmunity and allergic inflammation.Therefore, not only cancer, And generally infect, and the most chronically infected infection, autoimmunity and especially allergic inflammation are all is all Clinical disease related to the present invention.
IDO exists with 5 kinds of forms in people: IDO, IDOA, IDOB, IDOC and IDOLIKE (are also referred to as in the literature IDO2).IDO is the polypeptide of 403 amino acid residue length as disclosed in SEQ ID NO:1, and with SEQ ID NO:16 IDOLIKE be IDO preferred herein together.Other IDO is the most related to the present invention, although knowing little about it about them； They are identified as IDOA:SEQ ID NO:13, IDOB:SEQ ID NO:14 and IDOC:SEQ ID NO15 in this article. IDOLIKE expresses not as IDO widely, but as its relationship body, also expresses in antigen presenting dendritic cell, in institute State tryptophan catabolism in dendritic cell and drive immunologic tolerance.As IDO, IDOLIKE makes tryptophan catabolism, triggers The phosphorylation of translation initiation factor eIF2 α, and mobilize the translation of LIP, described LIP is immunomodulating transcription factor NF-IL6 A kind of inhibition hypotype (Popov & Schultze, 2008).The terms IDO is often referred to all above-mentioned IDO and they phases The sequence answered.
IDO has been identified as a kind of main immune modulatory molecules, and it is a part for several negative feedback mechanism, passes through These mechanism immunne response are under control.In this way, IDO also plays critical immune suppression function in cancer. In the research that the present invention is based on, examining the target whether IDO itself can serve as immunne response, it can be developed for exempting from Epidemic disease is treated, especially for the treatment of cancer.By taking " reversely immunity " mode, identify the HLA-A2 peptide in IDO albumen, Suffering from incoherent tumor type, i.e. the patient of melanoma, renal cell carcinoma and breast carcinoma detects spontaneous T to it Cellular responsibility.The t cell response of these naturally occurring readily uses after HLA/ peptide tetramer stimulates in vitro and passes through streaming Cell art and visible, and easily visible in direct isolated measuring.Additionally, confirm that IDO reaction-ive T cell is real clearly It it is the cytotoxic effect cell of peptide specific on border.In other words: it is (all that IDO specific T-cells dissolves separate sources effectively Such as melanoma, colon cancer, breast carcinoma and the AML blast cell of directly in vitro enrichment) IDO+ cancerous cell line.From trouble Have uncorrelated cancer types patient PBL in for the existence of spontaneous CTL response of peptide epitopes in IDO source and IDO Specific T-cells kills the cancerous cell of separate sources and illustrates the immunization therapy potentiality of IDO.Unique is following sending out Existing, i.e. IDO specific CTL identification also kills IDO+ maturation DC；Therefore, IDO specific T-cells can kill immunocompetence DC. Report in recent years has turned out IDO and is raised after maturation in people DC23,24.And, it is being envisaged for epidemic disease in cancer patient The DC of Seedling inoculation also observing, IDO expresses and this expresses and is held in place after s.c. (subcutaneous) injects25.Based on DC In treatment vaccine the expression of IDO via attract or induction FoxP3 (+) Treg keeps its critical clinical correlation.
The dual function (initial phase of suppression immunne response and effector phase) of IDO does not excludes each other；It practice, Likely both function both in specific tumor.Another effect that IDO plays may be high with the result of cancer immunotherapy Degree is relevant: as the anti-regulatory mechanism of inflammation-induced.Anti-regulation response is important in immune system, because they contribute to Limiting intensity and the degree of immunne response, otherwise immunne response may cause the dangerous infringement to host.But, exempt from regard to anticancer For epidemic disease treatment, anti-regulation response antagonism sets up the ability of strong immune response for tumor.It is only in response to immunity with regard to anti-regulation For the secondary event activated and cause, anti-regulation is different from tolerance.IDO is known to be lured by I type and II type interferon two types Leading, these interferon are likely found at immune activation and inflammation part26,27.It should be noted that to demonstrate at this and pass through Precincubation with IFN-γ adds the susceptibility killed that IDO reaction-ive T cell is caused by tumor cell.Similarly, report The systematicness in road costimulatory molecules 4-1BB (CD137) connects induction IDO28.Certain most of anti-cancer immunotherapy strategy is regardless of it Molecular target why, all for induction of immune activation and inflammation (such as, in position or the intra-tumor of vaccine).It is true that In acceptable toxicity range, reaching immune activation as much as possible is target；Therefore, anti-regulation is undesirable.With regard to this Speech, in blocking the melanoma and renal cell carcinoma patients that (that is, anti-CTLA-4 antibody) treat with CTLA-4, it has been reported that Relatedness between enterocolitis and tumor regression29.Therefore, autoimmune response clearly with clinical efficacy phase Close30,31.CTLA-4 blocking-up is received through suppressing the function of Treg to reduce the outer peripheral tolerance to autoantigen and increase T is thin Born of the same parents activate and mediate its antitumor and IRAE-inductive effect.
Owing to expressing the required effect of cell other immunotherapy methods of antagonism of IDO, such as by vaccination targeting Expressing the cell of IDO, adoptive T cell transfer or the immunostimulant aspect of the present invention (all these be all), therefore with other Anti-cancer immunotherapy is high Collaboration using.In the disclosure, it was demonstrated that the IDO epi-position that CTL limits can be answered widely In therapeutic vac-cination, and therefore there is important immunization therapy value.
Therefore it is an aspect of the invention to provide a kind of vaccine combination with the medicine acting on treatment clinical disease, It comprises IDO (IDO) or its immuno active polypeptide fragment.Described clinical disease can be cancer, and Another aspect of the present invention is prophylaxis of cancer, reduces risk or the treatment cancer forming cancer.On the other hand relate to the present invention's Vaccine combination and the use in conjunction of other drug such as immunotherapy medicaments and/or chemotherapeutics.Also have aspect relate to as The vaccine combination disclosed herein application in the treatment of viral and/or microbe-derived disease, and further relate to described Vaccine and other drug such as immunotherapy medicaments and/or antibiotic and/or the use in conjunction of antiviral agent.
Function congener
Sequence type
Wild type human IDO, i.e. the naturally occurring unmutated form of this protein, identifies in SEQ ID NO:1.This Invention is contained and is comprised following vaccine combination: IDO；The immunocompetence fragments of peptides of IDO；The compositions of the fragments of peptides of IDO, wherein At most two aminoacid are the most replaced；And/or the function congener of IDO, it has the sequence of at least 70% with SEQ ID NO:1 Row homogeneity.Term polypeptide fragment in this article be used for define amino acid residue any non-total length (as with SEQ ID NO:1 phase Than) chain, it is directed to the IDO as identified in SEQ ID NO:1, or synthesis is with same, or synthesis is to have with it There is the sequence iden of at least 70%.
Function congener can be defined as in sequence different from wild type IDO (such as wild type human IDO) but still The total length of IDO or the fragment of immunne response for the cell (such as cancerous cell and DC) expressing IDO can be induced.Thin at these The IDO expressed in born of the same parents can be wild type or endogenous mutant (the such as congenital mutant of induction during cell division Or sudden change etc.).Function congener can be with the mutant form or the alternative splicing form that are wild type IDO.In yet another aspect, The function congener of IDO is defined as described herein below.Function congener may be, but not limited to, and has in vitro introducing One or more sudden changes and/or one or more sequence deletion and/or the total length of interpolation or the recombinant forms of fragmentation IDO.
The function congener of IDO can be to show at least some sequence iden with SEQ ID NO:1 and have induction Any protein/polypeptide of ability to the immunne response of the cell expressing IDO.
Therefore, in a particular embodiment, the immunogenic activity fragments of peptides of the present invention is by as reflected in SEQ ID NO:1 Fixed IDO or 50 amino acid residues of its function congener, the most at most 45 amino acid residues, the most at most 40 ammonia Base acid residue, the most at most 35 amino acid residues, the most at most 30 amino acid residues, the most at most 25 aminoacid are residual Base, such as 18-25 continuous amino acid composition；Described function congener is the most at most three aminoacid, such as two amino Acid, the function congener that such as an aminoacid is the most replaced.
The most in another embodiment, the immunogenic activity fragments of peptides of the present invention is by from SEQ ID NO: The IDO of 1 or at most 25 amino acid residues of its function congener, the most at most 24 amino acid residues, the most at most 23 Amino acid residue, the most at most 22 amino acid residues, the most at most 21 amino acid residues, the most at most 20 aminoacid are residual Base, the most at most 19 amino acid residues, the most at most 18 amino acid residues, the most at most 17 amino acid residues, such as At most 16 amino acid residues, the most at most 15 amino acid residues, the most at most 14 amino acid residues, the most at most 13 Amino acid residue, the most at most 12 amino acid residues, the most at most 11 amino acid residues, such as 8-10 continuous amino acids Residue forms；Described function congener is the most at most three aminoacid, such as two aminoacid, and such as an aminoacid is Replaced function congener.Preferably, described peptide is identified in comprising to come SEQ ID NO:1 freely IDO or its function with At most 10 continuous amino acid residues of source thing, the most at most 9 continuous amino acid residues, such as 8 continuous amino acid residues, Such as from 7 continuous amino acid residues of IDO；Described function congener is the most at most three aminoacid, such as two ammonia Base acid, the function congener that such as an aminoacid is the most replaced.
The most in some embodiments, the peptide of the present invention is nonapeptide (comprising the peptide of 9 amino acid residues), and some ten Peptide (comprises 10 residues), and these can selected from following non-limiting group (be first peptide title, followed by sequence, Position in IDO sequence and SEQ ID NO.): IDO1:Q L R E R V E K L (54-62) (SEQ ID NO:2)； IDO2:F L V S L L V E I (164-172) (SEQ ID NO:3)；IDO3:T L L K A L L E I (195-203) (SEQ ID NO:4)；IDO4:F I A K H L P D L (41-49) (SEQ ID NO:5)；IDO5:A L L E I A S C L (199-207) (SEQ ID NO:6)；IDO6:V L S K G D A G L (320-328) (SEQ ID NO:7)；IDO7:D L M N F L K T V (383-391) (SEQ ID NO:8)；IDO8:V L L G I Q Q T A (275-283) (SEQ ID NO:9)；IDO9:K V L P R N I A V (101-109) (SEQ ID NO:10)；IDO10:K L N M L S I D H L (61-70) (SEQ ID NO:11)；IDO11:S L R S Y H L Q I V (341-350) (SEQ ID NO:12).Preferably Ground, the peptide of the present invention is IDO5:A L L E I A S C L (199-207) (SEQ ID NO:6)；IDO2:F L V S L L V E I (164-172) (SEQ ID NO:3)；And/or IDO6:V L S K G D A G L (320-328) (SEQ ID NO: 7).Most preferably, the vaccine combination of the present invention comprises and has IDO5:A L L E I A S C L (199-207) (SEQ ID NO:6) at least one peptide.
Other peptides of the present invention comprise (or more preferably consisting of) SEQ ID NO:1 IDO or with SEQ ID NO:1 There is the 4-120 of its function congener of at least 70% homogeneity, preferably 8-100, more preferably 10-75, even more preferably from 12-60, Even more preferably 15-40, such as 18-25 continuous amino acid, in described function congener, the IDO of SEQ ID NO:1 is extremely Many three aminoacid are the most replaced, delete or add, and such as two aminoacid is the most replaced, delete or add, or one Aminoacid is the most replaced, delete or add.
In one embodiment of the invention, IDO peptide comprises variant peptides.As used herein, statement " variant " refers to From base protein (suitably for people IDO) homology but the peptide different with the basic sequence obtaining them, difference is this sequence One or more aminoacid in row are replaced into other aminoacid.Suitable variant will have at most 6 amino acid replacements, example Such as at most 5 amino acid replacements, the most at most 4 amino acid replacements, the most at most 3 amino acid replacements, the most at most 2 ammonia Base acid is replaced, the most at most 1 amino acid replacement.
Suitable variant will share the sequence iden of at least 70% with the IDO of SEQ ID NO:1, and therefore, variant The preferably sequence with people IDO has at least 75% sequence iden, for example, at least 80% sequence iden, such as at least 85% sequence Row homogeneity, for example, at least 90% sequence iden, such as at least 91% sequence iden, for example, at least 91% sequence are same Property, such as at least 92% sequence iden, for example, at least 93% sequence iden, such as at least 94% sequence iden, such as At least 95% sequence iden, such as at least 96% sequence iden, for example, at least 97% sequence iden, such as at least 98% Sequence iden, such as 99% sequence iden.
Sequence iden can use the algorithm known to many and apply many different gap penalties to calculate.Sequence is same One property calculates relative to total length SEQ ID NO:1.Arbitrary sequence alignment tools, such as, but not limited to FASTA, BLAST or LALIGN, may be used for searching for congener and sequence of calculation homogeneity.Additionally, when in place, any known permutation matrix, all Such as, but not limited to, PAM, BLOSSUM or PSSM matrix, it is possible to use searching algorithm is applied.Such as, PSSM (remember by location specific Sub matrix (position specific scoring matrix)) can apply through PSI-BLAST program.Additionally, sequence Comparison can use point penalty that is a range of open for room and that extend to carry out.Such as, BLAST algorithm can use 5- Gap Opening Penalty in the range of 12, and the gap extension penalties in the range of 1-2.
Function equivalent can comprise further chemical modification such as ubiquitination, labelling (such as, use radionuclide, Various enzymes etc.), Pegylation (with Polyethylene Glycol derive) or by insertion (or being replaced by chemosynthesis) aminoacid (many Individual aminoacid) such as ornithine (during it is not present in people's albumen under normal circumstances), it is preferable, however, that function equivalent not comprising Learn and modify.
Any change made with the sequence of the IDO phase comparison amino acid residue of SEQ ID NO:1 is the most conservative puts Change.Those skilled in the art will know how to make and assess " guarding " amino acid replacement, by this conservative substitution, an amino Acid is replaced as another and has one or more common chemistry and/or the aminoacid of physical characteristic.Conservative amino acid replacement is relatively The functional of protein can not be affected.Aminoacid can be grouped according to common characteristic.Conservative amino acid replacement is pre- An aminoacid in the aminoacid of fixed group replaces to another aminoacid in identical group, and the aminoacid in the most predetermined group shows Show similar or essentially similar characteristic.
Therefore, in one embodiment of the invention, vaccine combination comprises the 8-50 of the IDO by SEQ ID NO:1 The polypeptide of the continuous sequence composition in individual amino acid whose scope, in preferred 8-10 or 20-25 amino acid whose scope, the most at most Three aminoacid are the most replaced, and preferred described displacement is conservative.
MHC
There is two kinds of MHC molecule；MHC I quasi-molecule and MHC II quasi-molecule.MHC I quasi-molecule is thin by CD8 T Born of the same parents identify, this cell is the main effects cell of adaptive immune response.MHC II quasi-molecule is mainly at antigen-presenting cell (APC) express on surface, most important of which seemingly dendritic cell.APC stimulates in Naive T cells, and immune system Other cells.They stimulate CD8 T cell and CD4 T cell.
In one embodiment, it is provided that new MHC I class-restricted peptides fragment, this fragments of peptides is by from SEQ ID 8-10 aminoacid composition of the IDO of NO1 or its function congener, wherein at most two aminoacid of SEQ ID NO1 by Displacement, is characterized by have at least one in several feature, and one of them is that to combine it with certain affinity be to limit Property the ability of I class HLA molecule, described affinity such as the half maximum alluvial (C by I class HLA molecule can be reached50Value) The amount of the peptide of (its at most 50 μMs) is measured, as being fitted to each other algoscopy (assembly binding by as herein described Assay) measure.This assembling algoscopy is based on HLA molecule after peptide is loaded to peptide transport protein deficient cells system T2 steady Fixed.Subsequently, the correct stable HLA heavy chain folded uses conformation dependent antibody mediated immunity precipitation, and carries out peptide combination quantitatively. The peptide of this embodiment comprise (or more preferably consisting of) SEQ ID NO1 IDO or wherein SEQ ID NO1 at most two At most 200 of its function congener that individual aminoacid is the most replaced, preferably up to 100, more preferably up to 50, the most more Preferably up to 25, even more preferably at most 20, the most at most 15, the most at most 10, such as 8-10 In the range of continuous amino acid.
This measures and provides screening candidate peptide and combine ability simple of specific HLA allele molecule with above-mentioned affinity Mode.In preferred embodiments, the fragments of peptides of the present invention is to have the C of at most 30 μMs50Value, the C of the most at most 20 μMs50 Value, including at most 10 μMs, at most 5 μMs and the C of at most 2 μMs50The fragments of peptides of value.
In another preferred embodiment, it is provided that the IDO of SEQ ID NO1 or the new MHC of its function congener II class-restricted peptides fragment, in described function congener, at most two aminoacid of SEQ ID NO1 are the most replaced, (this In literary composition also referred to as " peptide "), it is characterized by that there is at least one in several features described herein below.The peptide of this embodiment At most two aminoacid of the IDO that comprises (or more preferably consisting of) SEQ ID NO1 or wherein SEQ ID NO1 have been set to The 4-120 of its function congener changed, preferably 8-100, more preferably 10-75, even more preferably from 12-60, the most excellent Select 15-40, such as 18-25 continuous amino acid,.
It thus provides the new of the IDO of SEQ ID NO1 or its function congener has 8-10 amino acid whose MHC I Class-restricted peptides fragment and new there is 18-25 amino acid whose MHC II class-restricted peptides fragment, in described function homology In thing, at most two aminoacid of SEQ ID NO1 are the most replaced, it is characterized by have several features described herein below In at least one, one of them is that to combine be restrictive I class or the ability of II class HLA molecule to it.
In a particular embodiment, it is provided that fragments of peptides, it is that the MHCI class of at least one with following features limits Property peptide or MHC II class restricted peptides:
(i) can with such as measured by ELISPOT algoscopy at least 1/104The frequency of individual PBL is cancer patient's PBL group induces INF-γ and produces cell, and/or
(ii) can detect, at tumor tissues situ, the CTL reacted with described epitope peptide,
(iii) growth of IDO specific T-cells can be induced in vitro.
It is can to cause as by ELISPOT algoscopy (such as, embodiment herein below according to the preferred peptide of the present invention ELISPOT algoscopy described in 1) peptide of specific T-cells response that measures.Although some peptides do not combine I with high-affinity Class or II class MHC, but can still produce the t cell response as measured by ELISPOT.MHC can be combined with high-affinity Other peptides of I class or II class also produce the t cell response as measured by ELISPOT.Two kinds of peptide is according to the present invention Preferably peptide.
Therefore, it is to produce the specific T-cells as measured by ELISPOT algoscopy according to currently preferred peptide The peptide of response, the most every 108Individual cell, the most every 107Individual cell, the most every 106Individual cell, even more preferably from every 105 Individual cell, the most every 10450 specific spots of individual cell measurement.
It is can to cause in the individuality suffering to express the clinical disease that IDO is characterized according to the most preferred peptide of the present invention The peptide of cellullar immunologic response, described clinical disease is preferably cancer or infection, and most preferably cancer.
As it has been described above, HLA system code people's ajor histocompatibility (MHC) system.It is said that in general, MHC system controls one The inducement of plurality of properties: transplantation antigen, thymus dependent immunne response, some complement factor and some disease.More specifically, MHC encodes three kinds of different types of molecules, i.e. I class, II class and Group III molecule, and it determines the more common characteristic of MHC.At this In a little molecules, I quasi-molecule is so-called HLA-A, HLA-B and HLA-C molecule, and they are present in most of nucleated cell and blood coagulation On the surface of cell.
The feature of the peptide of the present invention is that they combine the ability of (being limited to) specific MHC I class HLA molecule.Therefore, exist In one embodiment, peptide is the peptide limited by MHC I class HLA-A molecule, and described I class HLA-A molecule includes HLA-A1, HLA- A2、HLA-A3、HLA-A9、HLA-A10、HLA-A11、HLA-Aw19、HLA-A23(9)、HLA-A24(9)、HLA-A25(10)、 HLA-A26(10)、HLA-A28、HLA-A29(w19)、HLA-A30(w19)、HLA-A31(w19)、HLA-A32(w19)、HLA- Aw33(w19)、HLA-Aw34(10)、HLA-Aw36、HLA-Aw43、HLA-Aw66(10)、HLA-Aw68(28)、HLA-A69 (28).The most also use simpler name, the most only use main number name, such as, HLA-A19 or HLA-A24 Replace HLA-Aw19 and HLA-A24 (49) respectively.In particular embodiments, the peptide of the present invention is limited to select free HLA- The MHC I class HLA species of the group of A1, HLA-A2, HLA-A3, HLA-A11 and HLA-A24 composition.In particular embodiments, The peptide of the present invention is limited to MHC I class HLA species HLA-A2 or HLA-A3.
In the most useful embodiment, the peptide of the present invention is the peptide limited by MHC I class HLA-B molecule, described I Class HLA-B molecule includes following any one: HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B13, HLA-B14, HLA- B15、HLA-B16、HLA-B17、HLA-B18、HLA-B21、HLA-Bw22、HLA-B27、HLA-B35、HLA-B37、HLA-B38、 HLA-B39, HLA-B40, HLA-Bw41, HLA-Bw42, HLA-B44, HLA-B45, HLA-Bw46 and HLA-Bw47.In the present invention Specific embodiments in, it is possible to combine the MHC I class HLA-B species of peptide of the present invention selected from HLA-B7, HLA-B35, HLA- B44, HLA-B8, HLA-B15, HLA-B27 and HLA-B51.
In the most useful embodiment, the peptide of the present invention is the peptide limited by MHC I class HLA-C molecule, described I Class HLA-C molecule include but not limited to following any one: HLA-Cw1, HLA-Cw2, HLA-Cw3, HLA-Cw4, HLA-Cw5, HLA-Cw6, HLA-Cw7 and HLA-Cw1.
In the most useful embodiment, the peptide of the present invention is the peptide limited by MHC II class HLA molecule, described II Class HLA molecule includes but not limited to following any one: HLA-DPA-1, HLA-DPB-1, HLA-DQA1, HLA-DQB1, HLA- All allele in DRA, HLA-DRB and these groups and HLA-DM, HLA-DO.
The known array of given specific HLA molecule can be combined by comparison thus be disclosed in peptide at specific site The predominance of minority related amino acid selects the peptide potentially with the ability combining specific HLA molecule.These are preponderated Amino acid residue referred to herein as " anchor residues " or " anchor residue motifs ".Based on can in enterable data base With the known array data found, by according to such relatively simple step, can obtain from IDO may be with concrete HLA The peptide that molecule combines.These representative example analyzed of a range of HLA molecule are given in the table below:
Table 2
The most in one embodiment, concrete anchor residues there is no for this position, but in preferred embodiments, This anchor residues is R or A.
Accordingly, as example, the nonapeptide potentially with the ability combining HLA-A3 has one of one sequence: Xaa-L- Y-Xaa-Xaa-Xaa-Xaa-Xaa-K, Xaa-L-Y-Xaa-Xaa-Xaa-Xaa-Xaa-Y；Xaa-L-Y-Xaa-Xaa-Xaa- Xaa-Xaa-F or Xaa-V-Y-Xaa-Xaa-Xaa-Xaa-Xaa-K (Xaa refers to any amino acid residue).In a similar fashion, may be used There is potentially the sequence of the ability combining any other HLA molecule with design.It is appreciated that those of ordinary skill in the art are by energy Enough more " anchor residue motifs " to the HLA Molecular Identification specified.
The peptide of the present invention can have such sequence, i.e. obtains the native sequences of its IDO.But, have higher Can be obtained by following method from such native sequences with the peptide of the affinity of any appointment HLA molecule: such as, based on Above-mentioned step, by displacement, deletes or adds at least one amino acid residue and modify this sequence, thus identify and refer to about this The anchor residue motifs of fixed HLA molecule.
Therefore, in useful embodiment, the polypeptide of the present invention includes such peptide, and its sequence comprises, in table Each of cited specific HLA allele, any one amino acid residue as pointed by table.
Therefore, the peptide of the present invention can be above-mentioned to comprise from any one in the peptide of the continuous sequence of IDO, wherein 1-10 Aminoacid in the range of aminoacid in the range of individual, preferably 1-5 are individual, the aminoacid in the range of more preferably 1-3, even more preferably Aminoacid in the range of 1-2, has replaced with another aminoacid even more preferably from 1 aminoacid, the most by this way, I.e. so that the grapplings one or more, the most all that this peptide comprises the HLA-A particular peptide specified as pointed out in upper table are residual Base.
The preferably example of HLA species includes, limits the HLA species of the preferred peptide of the present invention, including: select free HLA- The MHCI class HLA species of group of A1, HLA-A2, HLA-A3, HLA-A11 and HLA-A24 composition, more preferably this peptide by HLA-A3 or HLA-A2 limits.Alternatively, preferred HLA species include selecting free HLA-B7, HLA-B35, HLA-B44, HLA-B8, HLA- The MHC I class HLA-B species of the group of B15, HLA-B27 and HLA-B51 composition.
Identify that the mode of the polypeptide of the present invention comprises the following steps: to select specific HLA molecule, such as, go out with altofrequency Specify now the HLA molecule in colony, comparison analysis proceeded as above to identify " anchor residue motifs " in IDO albumen, Separate or build and comprise the one or more appropriately sized peptide in identified anchor residues and test gained peptide with such as At least 1/10 measured by ELISPOT algoscopy described in embodiment 14The frequency of individual PBL is in the PBL group of cancer patient Induction INF-γ produces celliferous ability.
In one aspect of the invention, it is provided that be longer than the IDO derived peptide of 8-10 amino acid residue.It is longer than 8-10 Amino acid whose polypeptide is processed into shorter length for combining HLA molecule by proteasome.Therefore, it is longer than 8-10 ammonia when using During the polypeptide of base acid residue, " length " polypeptides/proteins/protein fragments/variant of this IDO is added by proteasome in cytosol Work becomes a series of less peptide.Use the advantage of the longer polypeptide that can be processed into many different small peptides by proteasome be with A kind of 8-10 amino acid peptide limited by specific HLA type is compared, can be by a kind of peptide targeting more HLA type.
Unexpectedly, some in the peptide of the present invention are combined with MHC molecule with sufficiently high affinity so that displacement becomes (see Fig. 2) must be there is no need and easily use as antigen when they are presented at this.Preferably, the vaccine combination of the present invention Thing comprises following one or more: IDO albumen (SEQ ID NO:1), from its polypeptide fragment, from its variant, complete Length and the function congener of partial-length IDO, the continuous peptide of IDO and these function congener.It is highly preferred that vaccine combination Any one sequence enumerated in the sequence table comprising the disclosure.It is highly preferred that vaccine combination comprises peptide IDO5 (SEQ ID NO:6), IDO2 (SEQ ID NO:3) and/or IDO6 (SEQ ID NO:7).
The peptide of the present invention is noteworthy characterized by it and identifies or induction produces the ability of response T cell of INF-γ, described should Answer T cell, during i.e. specific recognition suffers from the individual PBL group of cancer and/or infection, APC or tumor/neoplastic cell (target Cell) on the cytotoxic T cell (CTL) of particular peptide.This activity is easily by by from individual PBL, APC or tumor Cell carries out ELISPOT mensuration and determines.Before the assay, stimulate this cell permissible by being contacted with peptide to be measured by cell to be measured It is favourable.Preferably, this peptide can with measured by ELISPOT algoscopy as used herein at least 1/104Individual PBL Frequency induction or identify produce INF-γ T cell.It is highly preferred that this frequency is at least 5/104Individual PBL, the most extremely Few 10/104Individual PBL, such as at least 50 or 100/104Individual PBL.
ELISPOT algoscopy represents a kind of powerful instrument monitoring IDO peptide-specific T-cell response.This paper sends out Existing major significance is that the peptide of the present invention is expressed and is combined with the HLA molecule on the APC of cancerous cell and/or expression IDO.This makes Obtain these cancerous cell to be prone to be destroyed by CTL, and highlight IDO immunity to anticancer and the potentially useful of infection.From black In the PBL of melanoma patient, the existence for the spontaneous CTL response of HLA restricted IDO derived peptide epi-position shows IDO immunity The immunization therapy potentiality of originality peptide.
In embodiments of the invention, the peptide of the present invention can induce in the individual PBL group suffer from clinical disease INF-γ produces cell, SEQ ID NO:(1,13,14,15 and/or 16 in described clinical disease) IDO or with SEQ ID NO1 has its function congener of at least 70% homogeneity and is expressed.Clinical disease is preferably cancer or/and infect, and Preferably cancer.
Source
As it has been described above, the peptide of the present invention derives from IDO or its fragment of SEQ ID NO:1,13,14,15 and/or 16, more Preferably, this peptide derives from the IDO of SEQ ID NO:1 and/or 16；Most preferably, this peptide derives from SEQ ID NO:1's IDO.Any IDO that the protein of this peptide can be from wherein expressing any animal species of this protein can be obtained.? In preferred embodiment, initial albumen is from mammalian species, including rodent, rabbit and primate such as People.Sequence based on selected protein, the peptide of the present invention passes through any suitable chemistry or the enzyme of protein initial substance Processing and obtain, described process obtains the most appropriately sized peptide, or it can pass through those of ordinary skill in the art Any conventional peptide symthesis step being familiar with synthesizes.Most preferably, when expressing the sequence of this protein in people, IDO albumen, Protein fragments, peptide, variant and/or these the function congener of any one derive from IDO.
Individual
The individuality treated with the vaccine combination of the present invention is the individuality suffering from clinical disease.Described individuality is preferably fed Breast animal species, and most preferably people.Individuality can be any age, youth or old, and can be that male is (male Property) or women (female).The clinical disease that this individuality suffers from can be neoplastic disease such as cancer, or infects such as microorganism Or virus infects, such as HIV.
Embodiment of the present invention provide for treating cancer, minimizing forms the risk of cancer, stablize cancer or prevention The vaccine of cancer.In another embodiment, present invention provide for treating and be derived from the disease of infection, reduce formation and be derived from The risk of disease, the disease being stably derived from infection or the prevention infected is derived from the vaccine of the disease of infection, and described infection is the most micro- Biological or virus infects.
More embodiment relates to the vaccine combination of a kind of clinical disease being characterized for treatment with expression IDO, It comprises SEQ ID NO:(1,13,14,15 and/or 16) IDO or with SEQ ID NO:(1,13,14,15 and/or 16) have Its function congener of at least 70% homogeneity or comprise the immunogen of continuous sequence of its function congener of described IDO or described Property active peptide fragments or encode the nucleic acid of described IDO or described fragments of peptides；And adjuvant.
Cancer
The vaccine combination of the present invention can be used to prevent clinical disease, the risk reducing formation clinical disease or treatment to face Bed disease.Preferably, clinical disease is relevant to the expression of IDO or is expressed as feature with IDO.IDO can be such as SEQ ID NOs:(1,13,14,15 and/or 16) in the IDO that identified in any one, and can be wild with any one in these Type form has at least 70% homogeneity but is necessarily the congener having function.It is therefore to be understood that the expression of IDO (should Expression is hnRNA, mRNA, precursor protein, the expression of albumen etc. processed completely) with the table in the individuality not suffering from clinical disease The level that reaches is identical or higher than the expression of the latter.In a preferred embodiment of the invention, this clinical disease is cancer.Cancer (malignant tumor) is such class disease, and one of which cell shows the uncontrolled growth (growth outside normal limit And division), invasion and attack (intrusion and destruction to adjacent tissue) and sometimes transfer (diffuse to its in health through lymph or blood His position) characteristic.The these three malignant nature of cancer makes it distinguish mutually with benign tumor, and benign tumor is self limiting, no Can invade or shift.Most of formation of cancer tumors, but some will not, such as leukemia.Term " cancer " meaning as used herein Disease (preneoplastic disease) before including any cancer, neoplastic disease and tumor.
The cancer be given as the example of the cancer can treated by using the vaccine of the present invention, control and/or prevent The non-limiting group of disease includes: colon cancer, breast carcinoma, cancer of pancreas, ovarian cancer, carcinoma of prostate, fibrosarcoma, myxosarcoma, Liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma (lymphangeosarcoma), lymphangioendothelial sarcoma (lymphangeoendothelia sarcoma), synovioma, mesothelium Tumor, ewing's sarcoma (Ewing ' s sarcoma), leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, gland Cancer, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma (cystandeocarcinoma), medullary carcinoma, a gas Pipe cancer, renal cell carcinoma, hepatocarcinoma, cancer of bile ducts, choriocarcinoma, spermocytoma, embryonal carcinoma, nephroblastoma, cervical cancer, testis are swollen Tumor, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioblastoma multiforme, neuroma (neuronomas), craniopharyngioma (craniopharingiomas), schwannoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma (craniopharyngioma), ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, brain Film tumor, melanoma, neuroblastoma, retinoblastoma, leukemia and lymphoma, acute lymphoblastic leukemia and Acute myelocytic polycythemia vera, multiple myeloma, Waldenstrom macroglobulinemia and heavy chain disease, urgency Property non-lymphocytic leukemia, chronic lymphocytic leukemia, chronic granulocytic leukemia, Hodgkin, non-Hodgkin′s Lymphoma, rectal cancer, urinary cancer, uterus carcinoma, oral cancer, skin carcinoma, gastric cancer, the cerebral tumor, hepatocarcinoma, laryngeal carcinoma, esophageal carcinoma, breast Adenoncus tumor, child's-null acute lymphoblastic leukemia (ALL), thymocyte cell type ALL, B cell type ALL, the white blood of acute myeloid Disease, myelomonocytic leukemia, acute megakaryoblastic leukemia, Burkitt lymphoma, acute myeloid leukemia, chronic myelogenous Leukemia and T cell leukemia, little and big nonsmall-cell lung cancer (small and large non-small cell lung Carcinoma), acute myeloblastic leukemia, germ cell tumor, carcinoma of endometrium, gastric cancer, incidence cancer, chronic pouring Bar chronic myeloid leukemia, hairy cell leukemia and thyroid carcinoma.
In preferred embodiments, clinical response can be caused in experimenter according to the vaccine combination of the present invention, Wherein the feature of this clinical response can be stable disease, and in preferred embodiments, the feature of this clinical response is permissible Be the feature of partial reaction or preferred this clinical response can be the complete incidence graph of cancer.Preferably, cancer is selected from following composition Group: melanoma, breast carcinoma, ovarian cancer, pulmonary carcinoma, cancer of pancreas, hematologic cancers (such as leukemia), colon cancer and nephrocyte Cancer.
In one aspect of the invention, vaccine combination can cause clinical response in individuality.An embodiment In, the feature of clinical response can be stable disease (no longer deteriorate or be in progress), in preferred embodiments, clinical response Feature can be the feature of partial reaction or preferred clinical response can be the complete incidence graph of cancer or infection.Clinical response can Determine with as described below.
In another aspect of the present invention, vaccine combination can cause clinical response, wherein this clinic in experimenter The feature of reaction is that the summation of the longest diameter of maximum target focus reduces.This reduction can as described below determine.
(all getting involved is represented up to all measurable focus of maximum 5 focuses of each organ and 10 focuses of total Organ) target focus and record and measurement when baseline should be designated.
Target focus should be based on its size (having the focus of longest diameter) and the fitness of its repeated measurement exactly (by imaging technique or clinical measurement) selects.
Calculate the summation of the longest diameter (LD) of all target focuses and be recorded as the total LD of baseline.The total LD of baseline will act as Characterize the object of reference of target tumor.
Every other pathological changes (or position of disease) should be identified as non-target focus, and should also be as remembering when baseline Record.The measurement of these focuses not necessarily, but the presence or absence of each focus should whole follow up a case by regular visits to period carry out record.
The evaluation of target focus
Reaction (CR) completely: the disappearance of all target focuses
Partial reaction (PR): the total LD of baseline is reduced at least 30% as object of reference, the LD summation of target focus
PD (PD): from treating and starting, minimum total LD of record is as object of reference, and the LD of target focus is total With increase at least 20%, or one or more new focus occurs
Stable disease (SD): from treatment start minimum total LD be object of reference, reduce be not enough to qualitative for PR or Increase is not enough to qualitative for PD
The evaluation of non-target focus
Reaction (CR) completely: all non-target foci disappearances and the normalization of Tumor Marker Levels
The disease (SD) of incomplete reaction/stable: the sustainable existence of one or more non-target focuses is or/and tumor-marker Thing level is maintained on normal limit
PD (PD): one or more new focus and/or the clear and definite progress of existing non-target focus occur
In one embodiment of the invention, the vaccine combination of any one of above-mentioned protein and/or polypeptide is comprised Can cause clinical response in experimenter, the feature of wherein said clinical response is the longest diameter summation of maximum target focus Reduce.
The vaccine combination of the expection present invention can cause when in the individuality being applied to suffer from the cancer expressing IDO for Exempting from of the cancer of the IDO expressing SEQ ID NO:1 or its function congener with SEQ ID NO:1 with at least 70% homogeneity Epidemic disease response.The vaccine combination of the present invention can induce generation in vaccinated individuality to have for cancerous cell, expresses IDO APC cytotoxic effect effector T cell and/or in experimenter inducing antigen-specific T cell in mesenchyma stroma of tumors Infiltration.
In addition to its ability causing immunne response in PBL group, it is also contemplated that the peptide of the present invention can be in situ i.e. in reality Body tumor tissues causes cytolytic immunne response.This can prove the most by the following: provides HLA-peptide complexes, example As, it is by multimerization and is provided with detectable label, and uses such complex for immunohistochemical staining with inspection In survey tumor tissues, the epitope peptide with the present invention has reactive CTL.Therefore, the further marked feature of the peptide of the present invention It is their ability to the CTL at the detection of tumor tissues situ and epitope peptide with reactivity.
It is also contemplated that the peptide of the present invention, the complex of HLA and peptide is caused to be presented on cell surface except it combines HLA molecule Beyond the ability of the target of epi-position or cytolytic T lymphocyte (this complex serve as again), can cause the other kinds of immunity should Answer, such as result in B cell response and/or the delayed hypersensitivity (DTH) of the antibody for complex.Latter type Immunne response is defined as the rubescent and palp scleroma at the injection site of the peptide of the present invention.
It is an object of the present invention to provide a kind of for prophylaxis of cancer, reduce and form risk or the treatment cancer of cancer Vaccine combination, it comprises SEQ ID NO:(1,13,14,15 and/or 16) IDO (IDO) or with SEQ ID NO:(1,13,14,15 and/or 16) have at least 70% homogeneity its function congener or comprise described IDO or The immunogenic activity fragments of peptides of the continuous sequence of its function congener described or encode the core of described IDO or described fragments of peptides Acid；And adjuvant.
Cancer therapeutic alliance
In some cases, by the Therapeutic Method of the present invention and other conventional cancer therapy such as chemotherapy, radiotherapy, use It is suitable that the treatment of the treatment of immunostimulation material, gene therapy, the treatment of use antibody and use dendritic cell is combined.
Owing in tumor cell, the expression increase of IDO causes immune suppression, therefore as by base disclosed by the invention Combining that immunotherapy in IDO is treated with cytotoxic chemotherapies and/or another kind of anti-cancer immunotherapy is treatment cancer Effective means.These medicines are referred to herein as " the second active component ".
Example with regard to chemotherapeutics relevant for the vaccine combination co-administered (sequentially or simultaneously) of the present invention Include, but are not limited to: all-trans-retinoic acid, actimide, azacitidine, azathioprine, bleomycin, carboplatin, capecitabine, Cisplatin, chlorambucil, cyclophosphamide, cytosine arabinoside, daunorubicin, Docetaxel, doxifluridine, doxorubicin, table Soft than star, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, irinotecan, Lenalidomide, Formyl tetrahydrofolic acid, chlormethine, melphalan, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, Lenalidomide (Revlimid), temozolomide, teniposide, thioguanine, valrubicin, vinblastine, vincristine, long fields for spring sowing Pungent and vinorelbine.In one embodiment, the chemotherapeutics for the drug combination with the present invention can itself be not assimilate Treat the associating of agent.Suitably combine and include FOLFOX and IFL.FOLFOX is to include 5-FU (5-FU), formyl tetrahydrofolic acid Associating with oxaliplatin.IFL treatment includes irinotecan, 5-FU and formyl tetrahydrofolic acid.
Another second active component can be inhibitors of kinases, in oncotherapy individually, simultaneously or be used in combination. Suitable inhibitors of kinases includes having shown that those (such as gefitinib (Iressa (Iressa)) with anti-tumor activity With Erlotinib (Erlotinib (Tarceva)) and these can be used in combination with peptide.Receptor tyrosine kinase inhibitors is also fitted Share work the second active component, all sunitinib malate (Sunitinib the most effectively treating renal cell carcinoma And Sorafenib malate).
More examples of the second active component are immunostimulation material, such as cytokine and antibody.These cytokines But can be not limited to selected from the group of the following composition: GM-CSF, I type IFN, interleukin-22 1, interleukin-22, interleukin 12 and white Interleukin 15.Antibody is preferably immunostimulation antibody such as anti-CD 40 or anti-CTLA-4 antibody.Immunostimulation material can also is that Can remove immunosuppressant cell (such as, regulatory T cells) or the material of the factor, described material can connect by e.g. E3 ubiquitin Connect enzyme.E3 ubiquitin ligase (HECT, RING and U-box protein) key molecule instrumentality as immune cell function goes out Existing, and each specificity Inhibitory molecules that all may be destroyed for Proteolytic enzyme by targeting and immune during participating in infecting The regulation of response.Also several HECT and RING E3 albumen are associated with induction and the maintenance of Immunological self-tolerance now: c- CbI, Cbl-b, GRAIL, Itch and Nedd4, the generation of each of which negatively regulatory T-cell somatomedin and propagation.
In one embodiment, the IDO that comprises of the present invention derives the vaccine combination of polypeptide and the second active component is all Such as immunostimulation material administering drug combinations.Immunologic stimulant is preferably interleukin such as IL-21 or IL-2, or chemotherapeutics.
Infect
As used herein word infects any kind of clinical disease relating to producing immunne response, such as inflammation, and Therefore infectious disease, chronic infection, autoimmune conditions and allergic inflammation are included.Therefore, infect, such as infect Property disease, chronic infection, autoimmune conditions and allergic disease, be all clinical disease related to the present invention, and And it is the most discussed below.Additionally, term infects and inflammation is used interchangeably herein.
Inflammation is that vascular tissue is anti-to the biology of the complexity of destructive stimulus such as pathogen, damaged cell or stimulus object Should.It is that organism is removed destructive stimulus and starts the protective measure that the agglutination of tissue is carried out.Inflammation can be divided Class is acute or chronic.Acute inflammation is the body primary response to destructive stimulus, and passes through blood plasma and leukocyte from blood In realize to the mobile increase in damaged tissues.The cascade of biochemical event is propagated and forms inflammatory reaction, relates to local vascular Various cells in system, immune system and damaged tissues.The inflammation extended, it is known that chronic inflammatory disease, causes being present in inflammation The Progressive symmetric erythrokeratodermia of the cell type at position migrates, and it is characterized in that tissue is by destroying while inflammatory process and healing.? In the case of each, IDO is expressed by immune cell such as APC, and therefore infection and inflammation are to pass through the present invention Using of vaccine combination can treat, prevent, or the clinical disease that its risk formed can reduce.Vaccine combination is excellent Selection of land comprises IDO albumen, protein fragments, its polypeptide derived or peptide or these the function congener of any one.
The example of the disease that inflammation related to the present invention is associated includes, but are not limited to: allergic inflammation, asthma, from Body immune disease, chronic inflammatory disease, chronic prostatitis, glomerulonephritis, allergy, infectious disease, inflammatory intestines disease, basin Chamber inflammatory inflammation, reperfusion injury, rheumatoid arthritis, graft-rejection and vasculitis.
Chronic infection and inflammation
Chronic inflammatory disease is especially relevant with the present invention.Chronic inflammatory disease is with concurrent activities inflammation, disorganization and to attempt to repair The pathological state being characterized again.The feature of chronically inflamed tissue is monokaryon immunocyte (mononuclear cell, macrophage, lymph Cell and plasma cell) infiltration, disorganization and attempt repair, the latter include blood vessel occur and fibrosis.
In acute inflammation, the removal of stimulation makes mononuclear cell (becoming macrophage when suitably activating) to Inflamed tissue Raise stopping, and existing macrophage leaves this tissue through lymphatic vessel.But in chronically inflamed tissue, stimulation is Persistent, and therefore maintain monocytic raising, existing macrophage is limited in situ, and stimulates huge biting The propagation (especially in atheromatous plaque) of cell.
It is an object of the present invention to provide a kind of for prophylaxis of chronic, reduce and form the risk of chronic inflammatory disease or control Treating the vaccine combination of chronic inflammatory disease, it comprises SEQ ID NO:(1,13,14,15 and/or 16) indoleamine 2, the double oxygenation of 3- Enzyme (IDO) or with SEQ ID NO:(1,13,14,15 and/or 16) there is its function congener or bag of at least 70% homogeneity The immunogenic activity fragments of peptides of the continuous sequence containing its function congener of described IDO or described or encode described IDO or described The nucleic acid of fragments of peptides；And adjuvant.
Infectious disease
The vaccine combination of the present invention can be used to prevent clinical disease, the risk reducing formation clinical disease or treatment to face Bed disease.In a preferred embodiment of the invention, clinical disease is infectious disease.Infectious disease can be by any infection Agent such as antibacterial, virus, parasite and/or fungus promote, these infectious agents can be induced in the individuality suffering from infectious disease The expression of IDO increases, it is preferable that infectious disease is chronic disease or is in the risk forming chronic disease.As invented the back of the body Described in scape, IDO express increase express IDO organism near by remove its tryptophan play to microorganism agent be Shi Zuoyong.But, when increase IDO induced expression suppression Treg cell active time, if this IDO express cell is APC, then Things turn out contrary to one's wishes for this mode.Therefore, it is an aspect of the invention to provide a kind of for treatment caused by infectious agent disease, improvement The vaccine combination of the disease that the stable and/or prevention of the disease that (alleviating seriousness) is caused by infectious agent is caused by infectious agent, It comprises IDO albumen, protein fragments, peptide and/or these the variant of any one.
Infectious disease can be caused by virus, and can use the vaccine combination of the present invention in for its treatment The virus disease of thing includes, but are not limited to following virus disease: HIV, AIDS, AIDS AIDS-related complex AIDS, chickenpox (Varicella), common cold, cytomegalovirus infection, colorado tick fever, dengue fever, ebola hemorrhagic fever, hand-foot-mouth disease Disease, hepatitis, herpes simplex, herpes zoster, HPV (human papilloma virus), influenza (Flu), lassa fever, measles, Marburg hemorrhagic fever, infectious mononucleosis, mumps, Norwalk viroid (Norovirus), spinal cord ash Matter inflammation, progressive multifocal leukoencephalopathy (Progressive multifocal leukencephalopathy), rabies, Rubella, SARS, variola (Variola), viral encephalitis, viral gastroenteritis, viral meningitis, viral pneumonia, western Buddhist nun Sieve river disease and yellow fever.Preferably, vaccine combination is applied to suffer from HIV/AIDS and the virus infection that may cause cancer Individual.The main virus relevant to human cancer is human papilloma virus, hepatitis B and hepatitis C virus, EB disease Poison, people's T lymph virus；Therefore it is of the present invention that the treatment infected as these viruses or the part as treatment are used Purpose.
The example that antibacterial related to the present invention infects includes, but are not limited to: anthrax, bacterial meningitis, botulism, Brucellosis, campylobacteriosis, cat scratch disease, cholera, diphtheria, epidemic typhus, gonorrhea, impetigo, legionellosis, Leprosy (Hansen's disease), leptospirosis, listeriosis, Lyme disease, melioidosis, rheumatic fever, MRSA infection, nocardiosis, Pertussis (Whooping Cough), the plague, pneumococcal pneumonia, psittacosis, Q heat, American spotted fever (RMSF), sramana Bacterium disease, scarlet fever, bacillary dysentery, syphilis, tetanus, trachoma, tuberculosis, tularemia, typhoid fever, typhus fever and urinary tract sense Dye.The vaccine providing treatment antibacterial infection and/or the infection of pre-bacteriological protection and/or minimizing to form the risk that antibacterial infects is the present invention A purpose.
Another aspect of the present invention is to provide for treating following disease and/or preventing following disease and/or reduce shape Become the vaccine combination of the risk of following disease: parasite infectivity disease such as, but is not limited to: african trypanosomiasis, ameba Disease, ascariasis, babesiasis, American trypanosomiasis, clonorchiasis sinensis, cryptosporidiosis, cysticercosis, diphyllobothriasis, dragon line Parasitosis, echinococcosis, enterobiasis, fascioliasis, fasciolopsiasis buski, filaricide, free living amoeba infection, giardia lamblia Disease, gnathostomiasis, hymenolepiasis, isosporiasis, kala azar, leishmaniasis, malaria, metagonimiasis, myiasis, Onchocerciasis, pediculosis, retrofection, scabies, schistosomicide, taeniasis, toxocariasis, toxoplasmosis, trichonematosis, rotation hair Parasitosis, trichuriasis, trichomoniasis and african trypanosomiasis；Fungal infectious disease, such as, but not limited to: aspergillosis, blastomycosis, beads Bacterium disease, ball spore bacterium disease, cryptococcosis, histoplasmosis, tinea pedis；Protein virus infectious disease, includes but not limited to: Transmissible spongiform Shape encephalopathy, mad cow disease, creutzfeldt-Jacob disease, Kuru fatal familial insomnia, and A Erpeisi syndrome；Therefore conduct It is the mesh of the present invention that the treatment of the infection that these parasites, fungus or Protein virus cause or the part as treatment are used 's.
Infectious disease therapeutic alliance
Further provide by using the vaccine combination according to the present invention permissible to the treatment of any infectious disease Be combined with other (second) active component give or with other treatment such as antibiotic therapy, chemotherapy, use immunostimulation The treatment of material, uses the treatment of dendritic cell, antiviral agent, antiparasitic etc. to combine and gives.
The example that can combine the second active component in the treatment of infectious disease with the vaccine of the present invention includes, But it is not limited to antibiotic.The terms antibiotic refers to have antibacterium, antifungal, antiviral and/or Antiparasitic Activity Material；Example related to the present invention includes, but are not limited to: amikacin, gentamycin, kanamycin, neomycin, Netilmicin, paromomycin, streptomycin, tobramycin, ertapenem, imipenum, meropenem, chloromycetin, fluoroquinolone Class, ciprofloxacin, Gatifloxacin, Gemifloxacin, grepafloxacin, levofloxacin, lomefloxacin, Moxifloxacin, norfloxacin, Ofloxacin, Sparfloxacin, trovafloxacin, glycopeptide, vancomycin, woods can amine, clindamycin, Macrolide/ketone lactone Class, azithromycin, clarithromycin, dirithromycin, erythromycin, cefadroxil, cefazolin sodium, cefalexin, cefalotin, head Spore woods, cefradine, cefaclor, cefamandole, cefonicid, cefotetan, cefoxitin, cefprozil, cephalo furan Pungent, Loracarbef, cefdinir, cefditoren, cefixime, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, head Spore cloth alkene, ceftizoxime, ceftriaxone, cefepime, single bacterium amine, aztreonam, nitro glyoxaline, metronidazole,Oxazolidone Class, Linezolid, penicillin, amoxicillin, amoxicillin/clavulante, ampicillin, sulbactam, bacampicillin, carboxylic benzyl XiLin, adjacent cloxacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxazacillin, benzylpenicillin, penicillin V, Piperacillin, piperacillin/Tazobactam Sodium, ticarcillin, ticarcillin/Clavulanate, link-typed circuit, quinupristin, Dalfopristin, sulfa drugs/sulfamethoxineAzoles, trimethoprim, Tetracyclines, demeclocycline, doxycycline, minot ring Element, tetracycline, pyrroles's antifungal drug clotrimazole, fluconazol, itraconazole, ketoconazole, miconazole, voriconazole, both sexes are mould Element B, nystatin, echinocandin, Caspofungin, MFG, ciclopirox, flucytosine, griseofulvin and terbinafine. That also relevant is antiviral agent such as vidarabine, acyclovir, gancyclovir and Wan Saiwei (valganciclovir), and nucleoside is similar to Thing reverse transcriptase inhibitors (NRTI): AZT (zidovudine), ddI (didanosine), ddC (zalcitabine), d4T (Si Tafu Fixed), 3TC (lamivudine), non-nucleoside reverse transcriptase inhibitor (NNRTI): nevirapine, Delavirdine, protease inhibitor: Saquinavir, ritonavir, indinavir, viracept see nelfinaivr, ribavirin, amantadine/rimantadine, Relenza (Relenza) and oseltamivir phosphate capsule (Tamiflu) but, that vertical, interferon of Pumi.
In one embodiment, the present invention relates to for the epidemic disease with at least one Antibiotic combination treatment infectious disease Seedling compositions, it comprises IDO derived protein, polypeptide and/or these function congener.Preferably, the vaccine combination of the present invention Thing is used for treating chronic infection, such as, HIV, and therefore with above-named antibiotic any one such as antiviral agent connection Close and use.
Autoimmune disease
Autoimmune is there is when himself ingredient (down to inframolecular level) can not be identified as self by organism Property disease, it causes the immunne response for himself cell and tissue.Any disease caused by such abnormal immune response Sick referred to as autoimmune disease, and related to the present invention.The example includes but not limited to: celiac disease (Coeliac Disease), type 1 diabetes (IDDM), systemic lupus erythematosus (sle) (SLE), dry syndrome, multiple sclerosis (MS), bridge are originally Thyroiditis, Graves disease (Graves ' disease), idiopathic thrombocytopenic purpura and rheumatoid arthritis (RA)。
It is an object of the present invention to provide a kind of for prevention of autoimmune diseases, minimizing formation autoimmunity disease Sick risk or the vaccine combination for the treatment of autoimmune disease, it comprises SEQ ID NO:(1,13,14,15 and/or 16) IDO (IDO) or with SEQ ID NO:(1,13,14,15 and/or 16) there is at least 70% homogeneity Its function congener or comprise its function congener of described IDO or described continuous sequence immunogenic activity fragments of peptides or Encode the nucleic acid of described IDO or described fragments of peptides；And adjuvant.
Autoimmune diseases is treated
The treatment being currently used for autoimmune disease is typically inhibitive ability of immunity, antiinflammatory or palliative.Non-exempt from Epidemic disease is treated, and the final result of the hormone replacement in the treatment of such as chronic lymphocytic thyroiditis or type 1 diabetes is their aggressive reaction.Diet Control to limit the order of severity of celiac disease.Steroid drugs or NSAID treatment limit the inflammatory symptoms of numerous disease.Immunoglobulin Intravenous formulations (IVIG) for chronic inflammation Demyelinating Polyneuropathy (CIDP) and Ge-bar two formula syndrome (GBS). More specifically immune modulating treatment, such as TNF α antagonist Embrel, have shown that and can be used for treating RA.These immunization therapies Relevant with the untoward reaction risk increased, such as it is prone to infect.
Observe based on these and have been developed for anthelmintic treatment, and it includes by special parasite intestinal nematodes (anthelmintic) Inoculation individuality.Two kinds of closely-related treatments are had to can use, with Necator americanus (being commonly called as ancylostome) or T.suis ovum at present (Trichuris Suis Ova) (being commonly called as Penis et testis sus domestica worm's ovum) is inoculated.There are some researches prove this mode in the many autoimmunity diseases for the treatment of Sick aspect is highly effective, and these diseases include crohn, ulcerative colitis, asthma, allergy, multiple sclerosis and slow Property inflammatory diseases.
In one embodiment, vaccine disclosed herein and the second active ingredient combination use, and described second activity becomes Point such as any one of the said medicine of autoimmune disease and treatment.
Allergic inflammation
Allergy is immune a kind of dysfunction, is also frequently referred to as idiosyncrasy (atopy).Abnormal anti- Should occur for the surrounding material being known as allergen；These reactions are acquired, predictable and quick.Strict next Saying, allergy is one of four kinds of forms of allergy, and referred to as I type (or anaphylactic type) allergy.It is characterized in that claiming For some leukocyte of mastocyte and basophilic granulocyte by the excessive activation of a type of antibody (being known as IgE), lead Cause inflammatory reaction extremely.Common allergy includes eczema, urticaria, pollinosis, asthma, food allergy and to stinging The reaction of the venom of thorn insecticide such as wasp and Apis.
Allergic inflammation is the important pathological characteristics of several deformity or medical conditions, including allergic asthma, spy Answering property dermatitis, allergic rhinitis and several ocular allergy disease.
It is an object of the present invention to provide a kind of for preventing allergic inflammation, reducing formation abnormal reactive inflammation Disease or treatment allergic inflammation vaccine combination, it comprises SEQ ID NO:(1,13,14,15 and/or 16) indole Amine 2,3-dioxygenase (IDO) or with SEQ ID NO:(1,13,14,15 and/or 16) there is its merit of at least 70% homogeneity Can congener or comprise its function congener of described IDO or described continuous sequence immunogenic activity fragments of peptides or coding institute State the nucleic acid of IDO or described fragments of peptides；And adjuvant.
Allergic inflammation therapeutic alliance
Two kinds for the treatment of can be used for treat allergic inflammation: pharmacotherapy and immunotherapy.
Pharmacotherapy, is to use agonist drug to block the effect of allergy medium, or stops the activation of cell and de- Granule process.These include antihistaminic, cortisone, dexamethasone, hydrocortisone, epinephrine (adrenaline), Theophylline, sodium cromoglicate, anti-leukotriene medicine, such as montelukast (Singulair) or zafirlukast (Accolate)；Anticholinergic, subtract and fill Blood agent, mast cell stabilizers, and also generally use and think other compounds of infringement Eosinophil Chemotaxis.
Immunotherapy is to subtract quick or desensitization treatment, and wherein the target of the individual dosage inoculated progressively with gradual increase becomes Ying Yuan.The immunotherapy of the second form includes intravenous injection anti-ig E monoclonal antibody.The third type Sublingual immunity Therapy is Orally administered therapy, it makes use of non-pathogenic antigen such as food and the oral tolerance of resident antibacterial Property.
In one embodiment, vaccine disclosed herein and the second active ingredient combination use, and described second activity becomes Point such as any one of the said medicine of allergic inflammation and treatment.
Pharmaceutical composition
The present invention relates to can to treat in individuality and express relevant clinical disease to IDO, reduce this clinical disease of formation Risk and/or prevent the pharmaceutical composition of this clinical disease；In other words, term vaccine and pharmaceutical composition in this article may be used Exchange and use.Vaccine/the pharmaceutical composition of the present invention can be " traditional " vaccine combination, and it is many that it comprises antigen such as albumen Peptide and/or nucleic acid molecules.They can also is that bag celliferous composition forms, described cell be such as derived from this individuality with after The modified cells of processing, or comprise the compositions of complex molecule such as antibody or TCR.
Generally, vaccine is material or the compositions that can induce immunne response in individuality.Under said composition can comprise One or more of row: " active component " such as one or more antigens (such as, protein, polypeptide, peptide, nucleic acid etc.), except Other elements also comprise the nucleic acid construct of one or more antigens, and (such as, the APC of load, for adoptive transfer for cell The T cell of (adoptive transder aso.)), complex molecule (antibody, TCR and MHC complex etc.), carrier, assistant Agent, and pharmaceutical carrier.Below, each component of the vaccine combination according to the present invention it is disclosed more closely in.
The vaccine combination of the present invention is energy when being applied to suffer from cancer and/or infecting (causing the expression of IDO) individual Enough cause for expressing the IDO of SEQ ID NO:1 or with SEQ ID NO1, there is its function congener of at least 70% homogeneity Cancer, the immunne response of DC or APC.In preferred embodiments, this clinical disease is cancer.The vaccine combination of the present invention Thing can induce generation in vaccinated individuality to be had for expressing the cancerous cell of IDO, the cytotoxic effect of APC and DC Effector T cell and/or inducing antigen-specific T cell infiltration in tumor stroma in experimenter.
Antigen and other active components
Vaccine combination based on protein/polypeptide
The peptide of the present invention combines (seeing Fig. 2) with unexpected high affinity, and easy when they are presented at this As antigen.Preferably, the vaccine combination of the present invention comprises one of the following or multiple: IDO albumen (SEQ ID NO: 1), from its polypeptide fragment, from its variant, the function congener of total length and partial-length IDO, the continuous peptide of IDO and These function congener.It is highly preferred that vaccine combination comprise the disclosure sequence table in any one sequence of enumerating.Very Preferably, vaccine combination comprises peptide IDO5 (SEQ ID NO:6), IDO2 (SEQ ID NO:3) and/or IDO6 (SEQ ID NO:7).
In the vaccine combination of the present invention, the selection of antigen will depend upon which the parameter that can be determined by those skilled in the art. As already mentioned, each of different peptides of the present invention all by specific HLA molecular presentation on cell surface.As This, if experimenter to be treated classifies about HLA phenotype, then select this specific HLA molecule of known combination one or Multiple peptide.Alternatively, based on specifying the dominance of various HLA phenotypes in colony to select purpose antigen.As an example, HLA-A2 It is the most dominant phenotype in Caucasian (Caucasian population), and therefore, containing the peptide combining HLA-A2 Compositions will major part this colony in be effective.Additionally, the antigen/peptide of the present invention can be according to the anchor be given in table 2 Determine residue motif to carry out modifying to strengthen the combination with specific HLA molecule.
The compositions of the present invention can also be containing the combination of two or more IDO derived peptide, and every kind of peptide divides from different HLA Son specifically interacts to cover the major part of target colony.Accordingly, as example, pharmaceutical composition can be containing being subject to Combining of peptide that HLA-A molecule limits and the peptide that limited by HLA-B molecule, such as, include and the advantage of HLA phenotype in target colony Spend those corresponding HLA-A and HLA-B molecules, such as, such as HLA-A2 and HLA-B35.It addition, said composition can comprise The peptide limited by HLA-C molecule.
In the case of vaccine based on peptide, epi-position can be used with the form of " MHC-standby (MHC-ready) ", its energy Enough by presenting independent of the exogenous load of antigen uptake and being processed by host antigen-presenting cells.The peptide of the present invention Comprise the two kinds of peptides being in the longer form that short " MHC-is standby " form is processed by proteasome with needs, thus provide more Complicated vaccine combination, it can be with targeting kinds of tumors antigen.The different HLA groups of vaccine targeting are the most, then vaccine is in difference Colony in the probability that works the highest.
The present invention relates to a kind of vaccine combination as medicine in preferred embodiments, and it comprises SEQ ID NO: The IDO (IDO) of 1 or there is its function congener or bag of at least 70% homogeneity with SEQ ID NO:1 The immunogenic activity fragments of peptides of the continuous sequence containing its function congener of described IDO or described or encode described IDO or described The nucleic acid of fragments of peptides；And adjuvant.This vaccine combination can be used to treat clinical disease, prevention clinical disease in individuality Or reduce the risk relevant to clinical disease.
Polyepitope vaccines compositions
The invention still further relates to high immunogenicity polyepitope vaccines.Preferably, such vaccine should be designed to promote Deliver while the IDO derived peptide being suitable for, optionally in combination with other suitable peptide and/or adjuvant, as described below.The present invention Including such polyepitope vaccines, it comprises IDO derived peptide, is not belonging to or not from the egg of IDO optionally in combination with other White matter or fragments of peptides and/or adjuvant, as described below.The key factor ordering about the vaccine that exploitation has more complicated composition is target To the hope of kinds of tumors antigen, such as, contained by design packet or CTL and T of coding selection meticulouslyhThe set of cell epitope Vaccine.Therefore the present invention relates to the vaccine combination comprising the IDO epi-position of I class and the restriction of II class in one aspect.
Therefore the peptide of the present invention comprises and is in short " MHC-is standby " form (I class is restricted) and needs are added by proteasome Two kinds of peptides of the longer form (II class is restricted) of work.Therefore, the compositions according to the present invention can be as defined above The polyepitope vaccines of the epi-position that the epi-position comprising the restriction of I class and/or II class limit provides.
Vaccine combination based on nucleic acid
Vaccine combination according to the present invention can comprise protein or its immunologic competence fragments of peptides that coding belongs to IDO Nucleic acid.Therefore described nucleic acid can encode any one of above mentioned protein and fragments of peptides.Nucleic acid can be such as DNA, RNA, LNA, HNA, PNA, preferred nucleic acid is DNA or RNA.
The nucleic acid of the present invention may be embodied in any suitable carrier, such as expression vector.A large amount of carriers can utilize, And technical staff will can be that concrete purpose selects useful carrier.Carrier it may be that such as, plasmid, cosmid, virus Grain or the form of artificial chromosome.Suitable nucleotide sequence can be inserted in carrier by multiple method, and such as, DNA is permissible Technology well known in the art is used to be inserted in suitable one or more restricted enzyme nucleic acid sequences.Dig up the roots according to this Beyond bright nucleotide sequence, carrier can additionally comprise signal sequence, replication origin, one or more marker gene, enhancing One or more in sub-element, promoter and transcription terminator.Carrier can also comprise other sequence, such as strengthens Son, poly-A tail, joint, polylinker, operation joint, multiple clone site (MCS), STOP codon, internal ribosome enter Angle of striking (IRES) and for integrate host's homologous sequence or other elements determined.For the method transforming nucleic acid construct It is well known in the art (to see, e.g., Molecular Cloning:A Laboratory Manual (molecular cloning: real Test room guide), Sambrook etc., editor, Cold Spring Harbor Laboratory, second edition, Cold Spring Harbor, N.Y., 1989).Carrier is preferably expression vector, comprises the nucleic acid being operatively connected with modulability nucleotide sequence, This modulability nucleotide sequence guides its expression in suitable cell.Described modulability nucleotide sequence generally should guide Expression in mammalian cell, preferably people's cell, more preferably antigen-presenting cell, this is included within the scope of the invention.
In a preferred embodiment, carrier is viral vector.This carrier can also is that bacteria carrier, is such as attenuated Bacteria carrier.Attenuation bacteria carrier can be used to induce lasting mucosal immune response at infection site and make it persistently deposit ?.Different recombinant bacterias can serve as carrier, and such as, bacteria carrier can select free Salmonella, Lactococcus and Li Si The group of special Pseudomonas composition.Generally, the induction of immunity to heteroantigen HPV16 L1 or E7 can be shown, have in mice Strong CTL induction and tumor regression.Carrier can additionally comprise coding T cell stimulates the nucleic acid of polypeptide.
The APC of load
In useful embodiment, thin by MHC I class or II quasi-molecule being supported on from individual antigen presentation On born of the same parents (APC), by separating PBL and by the peptide incubation of described cell Yu the present invention from this individuality, then this cell infusion is returned This individuality, or by separating precursor APC from this individuality and using cytokine and antigen that this cell is divided into full-time APC, Then this cell infusion is returned this individuality, thus causes the immunogenicity for Cancerous disease to answer by using the peptide of the present invention Answer.
Therefore it is an aspect of the invention to provide such vaccine combination, it comprises IDO or its immunologic competence peptide Fragment or encoding said proteins or the nucleic acid of described immunologic competence fragments of peptides.Antigen-presenting cell can be can by antigen in It is handed to any cell of T cell.Preferably antigen-presenting cell is dendritic cell.Dendritic cell (DC) can according to any suitably Testing program, testing program the most as described below, prepare in Therapeutic Method and use.Those skilled in the art will manage Solving, this testing program can be altered for having different HLA type and suffering from the individual use of various disease.
Dendritic cell (DC) can be with 50 μ g/ml HLA-restricted peptides (synthesizing with GMP mass) pulse at 37 DEG C (pulse) 1h, peptide and 5x106Cell, the 1st day and the 14th day subcutaneous administration, is used for the most every 4 weeks, laggard 5 inoculations The Leukapheresis that row is other.The generation of DC and quality control for clinical practice can be substantially such as Nicolette etc. (2007) carry out described in.
Therefore, in one embodiment of the invention, treatment suffer from IDO be expressed as feature clinical disease The method of body, the most wherein said clinical disease is cancer or infection, and the method is wherein to pass through peptide in vitro It is presented to the antigen-presenting cell (APC) of this individuality, then the APC thus processed is injected back in this individuality and use this peptide Method.There is at least two alternate ways carrying out the method.A kind of alternate ways is to separate APC from individuality, and by MHC I class Molecule and this peptide incubation (load).Load MHC I quasi-molecule refers to make with the MHC special to this peptide APC with peptide incubation The APC of I quasi-molecule will be in conjunction with this peptide and therefore, it is possible to be presented to T cell.Subsequently, APC is injected in individuality again. Another alternate ways depends on the discovery made in dendritic cell field of biology in recent years.In the case, mononuclear cell (for dendritic cell precursor) separates from individuality, and becomes full-time APC by using cytokine and antigen to break up in vitro (or dendritic cell).Then, the DC of external generation with this peptide pulse and is injected in this individuality.
Adoptive immunotherapy/adoptive transfer
One importance of the present invention relates to In vitro culture IDO specific T-cells and by they adoptive transfers to individual. Adoptive transfer refers to that the immune real composition having been able to produce specific immune response is directly transferred to individual by doctor Body.
It is an object of the present invention to provide IDO specific T-cells, it may be used for, such as adoptive transfer.Comprising can The separation T cell of the φt cell receptor of specific binding IDO peptide/MHC I class or IDO peptide/MHC II class complex can be adopted Being transferred to individuality, the T cell that described T cell preferably expands the most in vitro, wherein said IDO peptide can be herein above Any one of the IDO peptide mentioned.Known to the method for amplification in vitro T cell for those of ordinary skill is.The invention still further relates to Therapeutic Method, the method includes comprise can the T cell of φt cell receptor of specific binding MHC restricted IDO peptide complexes Being applied to the individual people such as suffering from Cancerous disease, wherein said IDO derived peptide can be any one of above-mentioned IDO peptide.In addition The present invention relates to comprise can the T cell of φt cell receptor of specific binding IDO or its fragments of peptides in preparation for treating cancer Or the application in the medicine infected.Autologous T cells transfer can be carried out described in (1995) substantially such as Walter etc..
TCR shifts
The most in another embodiment, such T cell can be illuminated to control at individuality before adoptive transfer In propagation.Can be by the specificity (Engels etc., 2007) of tcr gene transfer transformation T cell.This can by with The T cell of IDO peptide specific is transferred in individuality.Generally, T cell application in adoptive immunotherapy is attractive, Because it allows to expand T cell in without tumor or virus-free environment, and analyzes T cell function before infusion.TCR base Because of T cell (T cell such as converted by the expression construct the guiding allos TCR to express) application in adoptive transfer modified There is compared with transfer with T cell system several advantage: the generation that (i) is redirected T cell is the most applicable.(ii) can select Select or set up high-affinity or the TCR of high affinity, and be used for transforming T cell.(iii) use codon optimized Or Mus source (murinized) TCR can generate high-affinity T cell, it is allowed to the more preferably surface expression of stable TCR.Logical Cross φt cell receptor (TCR) gene transfer transformation T cell specificity to enter described in (2006) substantially such as Morgan etc. OK.
TCR transfects
The TCR having known antitumor reactive can be introduced in primary human T lymphocyte hereditarily.Encode from The TCR α of tumor-specific CTL clone and the gene of β chain can transfect to primary T cells and reprogramming T in this way The specificity for tumor antigen of cell.TCR RNA is by electroporation transfection to (Schaft etc., 2006) in PBL.Alternative Ground, T cell can use retroviral vector to pass through tcr gene transfer and provide with new specificity (Morgan etc., 2006). But, the provirus from retroviral vector may be incorporated in the genome of transfectional cell randomly and interference subsequently is thin Intracellular growth.This shortcoming is overcome by the RNA electroporation T cell of coding TCR, because RNA is only temporarily present in transfectional cell, And (Schaft etc., 2006) can not be integrated in genome.Additionally, the transfection of cell is routinely used in the lab.
Adjuvant and carrier
Vaccine combination according to the present invention preferably comprises adjuvant and/or carrier.Available adjuvant and the example of carrier It is provided below.Therefore IDO albumen, polypeptide fragment, variant therefrom or peptide can be combined in this with adjuvant and/or carrier In the compositions of invention.
Adjuvant is its mixing in vaccine combination to increase or change in other manners to exempt from IDO or its fragments of peptides Any material of epidemic disease response, with further reference to following.Carrier is supporting structure, such as polypeptide or polysaccharide, its can with IDO or its Fragments of peptides combines and it contributes to the presenting of peptide of the especially present invention.
Many peptides of the present invention are relatively small molecules, and therefore it may need peptide and many kinds of substance such as adjuvant And/or carrier is combined in compositions as described herein to produce vaccine, immunogenic composition etc..Adjuvant, from broadly Definition, is the material promoting immunne response.The comprehensive discussion of adjuvant provides at Goding, Monoclonal Antibodies: In the 61-63 page of Principles & Practice (monoclonal antibody: principle and put into practice) (second edition, 1986).Goding Notice, when purpose antigen has low-molecular-weight, or during immunogenicity difference, it is recommended that be combined with immunogenic carrier.Such The example of carrier molecule includes keyhole limpet hemocyanin, bovine serum albumin, ovalbumin and poultry immunity globulin (fowl immunoglobulin).Have been proposed that the adjuvant that multiple saponin extract also is used as in immunogenic composition.Carry Go out use a kind of known to cytokine granulocyte-M-CSF (GM-CSF) as adjuvant (WO97/ 28816)。
Carrier can exist independent of adjuvant.The function of carrier can be the molecular weight such as increasing particularly fragments of peptides Thus increase its activity or immunogenicity, imparting stability, increase biologic activity or increase serum half-life.Additionally, carrier IDO albumen, polypeptide, its variant or fragments of peptides presenting to T cell can be assisted.Carrier can be that those skilled in the art know Any suitable carrier, such as, protein or antigen-presenting cell.Carrier protein may be, but not limited to, and keyhole relative blood is blue Albumen, serum albumin such as transferrins, bovine serum albumin, human serum albumin, Elityran or ovalbumin, exempt from Epidemic disease globulin, or hormone, such as insulin or Palmic acid.For the immunity of people, carrier must be that the acceptable physiology of people can Accept carrier and be safe.But, in one embodiment of the invention, tetanus toxoid and/or diphtheria class Toxin is suitable carrier.Alternatively, carrier can be glucosan such as agarose (sepharose).
Therefore, the IDO albumen during one aspect of the present invention is present in compositions, polypeptide fragment, change therefrom Body or peptide and carrier albumen the most above, or antigen-presenting cell the most such as dendritic cell (DC) combination.
Adjuvant can be selected from the group of following composition: AlK (SO4)2、AlNa(SO4)2、AlNH4(SO4), titanium dioxide Silicon, Alumen, Al (OH)3、Ca3(PO4)2, Kaolin, carbon, aluminium hydroxide, muramyldipeptide, N-N-acetylmuramyl-L-threonyl- (CGP 11687 is also referred to as D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine Nor-MDP) and the different glutamy of N-acetylmuramyl-L alanyl-D--ALANINE-2-(1 ' 2 '-two palmityl-sn-glycerol- 3-hydroxyl phosphinylidyne epoxide) ethamine (CGP 19835A, also referred to as MTP-PE), in 2% Squalene/Tween-80.RTM emulsion RIBI (MPL+TDM+CWS)；Lipopolysaccharide and various derivant thereof, the completeest including lipid A, Freund's complete adjuvant (FCA), Freund Full adjuvant, Merck adjuvant 65 (Merck Adjuvant65), polynucleotide (such as, poly IC and poly AU acid)；From knot The wax D of core mycobacteria；Become with Brucella (genus Brucella's) coryne bacterium parvum, pertussis Boulder spy bacterium The material found in Yuan；Titermax, ISCOMS, Quil A, ALUN (see US 58767 and 5,554,372), lipid A derive Thing, cholera toxin derivant, HSP derivant, LPS derivant, synthetic peptide substrate or GMDP, interleukin-11, interleukin-22, Montanide ISA-51 and QS-21.Preferred adjuvant for the present invention includes adjuvant based on oil/surfactant such as Montanide adjuvant (available from Seppic, Belgium), preferably Montanide ISA-51.Other preferred adjuvants are based on carefully The adjuvant of bacterium DNA, such as includes the adjuvant of CpG ODN sequence.Other preferred adjuvants also have assistant based on virus dsRNA Agent, such as poly I:C.Imidazoquinolie (imidazochinilines) is also another example of preferred adjuvant.Most preferably Adjuvant is suitable for the adjuvant that people uses.
Montanide adjuvant (all available from Seppic, Belgium) can be selected from the group of the following composition: Montanide ISA-51、Montanide ISA-50、Montanide ISA-70、Montanide ISA-206、 Montanide ISA-25、Montanide ISA-720、Montanide ISA-708、Montanide ISA-763A、 Montanide ISA-207、Montanide ISA-264、Montanide ISA-27、Montanide ISA-35、 Montanide ISA 51F, Montanide ISA 016D and Montanide IMS, be preferably selected from by Montanide ISA- 51, the group of Montanide IMS and Montanide ISA-720 composition, is more preferably selected from being made up of Montanide ISA-51 Group.Montanide ISA-51 (Seppic, Inc.) is adjuvant based on oil/surfactant, lives in the most different surfaces Property agent and nonmetabolizable mineral oil, metabolizable oil or the combination of both mixture.They with comprise IDO or its fragments of peptides Prepared by aqueous solution, as emulsion.Surfactant is mannide oleate.QS-21(Antigenics；Aquila Biopharmaceuticals, Framingham, MA) it is highly purified water-solubility saponin, it operates as aqueous solution. QS-21 and Montanide ISA-51 adjuvant can provide in aseptic disposable bottle.
Known to cytokine GM-CSF be another preferred adjuvant of the present invention.GM-CSF is used for ten as adjuvant Year, and may be preferred that the GM-CSF as described in WO 97/28816.
The desired function of adjuvant that can be used according to the invention is enumerated in the following table.
Table 2: adjuvant effect mode
Source: Cox, J.C., and Coulter, A.R. (1997) .Vaccine15,248-56.
Vaccine combination according to the present invention can comprise more than one adjuvant.Additionally, the present invention includes a kind for the treatment of Property compositions, it also comprises any adjuvant substance and/or the carrier including above-mentioned any one or a combination thereof.It is also contemplated that IDO egg In vain, its variant or fragments of peptides and adjuvant can separate administration in any suitable order.Preferably, the vaccine combination bag of the present invention Containing Montanide adjuvant such as Montanide ISA51 or Montanide ISA720 or GM-CSF adjuvant.
Therefore, the present invention includes a kind of therapeutic composition, and it also comprises the assistant including above-mentioned any one or a combination thereof Agent material.It is also contemplated that antigen (that is, the peptide of the present invention) and adjuvant can simultaneously or in any suitable order separate administration.
Dosage and administration
In pharmaceutical composition, the amount of the immunogenic peptide of the present invention can change according to special application.But, peptide The single dose of compositions is preferably from about all such as from about 100 μ g-about 1000 μ g of 10 μ g-about 5000 μ g, more preferably from about 50 μ g-about 2500 μ g In any value.Administering mode includes Intradermal, subcutaneous and intravenous administration, with the implantation etc. of the form of timed release preparations.? Include as known in the art any and all form of medication herein.Also include being suitable for as is generally known in the art preparing injectable Any and all regular dosage form of immunogenic peptide compositions, such as lyophilized form and solution, suspension or emulsion form, if needed Want, containing conventional pharmaceutically acceptable carrier, diluent, preservative, adjuvant, buffer components etc..
Pharmaceutical composition can use any conventional methods well known by persons skilled in the art preparation and use.In reality Executing in example 3-5, the limiting examples and the such vaccine that give the preparation of the vaccine combination according to the present invention are administered Limiting examples.It will be understood by those skilled in the art that this test method can easily vary to be suitable for described herein Any one vaccine combination.In more embodiments of the present invention, the pharmaceutical composition of the present invention can be used for treatment to be suffered from The individuality of the clinical disease (such as cancer and infection) of feature it is expressed as with IDO.
The immanoprotection action of the compositions of the present invention can use several method well known by persons skilled in the art to come really Fixed.Successfully immunne response can also be by the generation of DTH reaction after immunity and/or detection this vaccine combination of specific recognition The antibody of one or more peptides determine.
Vaccine combination according to the present invention can be applied to individuality with therapeutically effective amount.Effective dose can according to multiple because of Element and change, situation, body weight, sex and the age that these factors are such as individual.Other factors include administering mode.
Pharmaceutical composition can be supplied to individuality by number of ways, the most subcutaneous, locally, per os and intramuscular.Medicine group The administration oral administration of compound or parenteral realize.The method of parenteral delivery includes local, intra-arterial (directly into tissue), flesh In interior, subcutaneous, marrow, in sheath, in ventricle, intravenous, intraperitoneal or intranasal administration.The present invention also has such purpose: provide Suitably local, oral, whole body and parenteral drug preparation are for using in the prevention of vaccine combination and the method for the treatment of.
Such as, vaccine combination can be with such as tablet, capsule (respectively including time delay release and extended release preparation), ball The peroral dosage form of agent, powder, granule, elixir, tincture, solution, suspension, syrup and emulsion, or used by injection.Equally Ground, they can also with Intravenous forms (injecting and infusion two kinds), intraperitoneal form, subcutaneous form, with and without suction The localized forms or the intramuscular form that stay (occlusion) are used, well-known to the ordinarily skilled artisan in all uses pharmaceutical fields Form.Effective but the vaccine comprising any one compound as herein described of avirulent amount can serve as preventive or treatment Agent.Also include any and all regular dosage form being suitable for preparing injectable immunogenic peptide compositions as is generally known in the art, all Such as lyophilized form and solution, suspension or emulsion form, if it is desired, containing conventional pharmaceutically acceptable carrier, diluent, prevent Rotten agent, adjuvant, buffer components etc..
The preferred modes of the vaccine combination according to the present invention includes, but are not limited to Formulations for systemic administration, the most quiet In arteries and veins or subcutaneous administration, intradermal administration, intramuscular administration, intranasal administration, oral administration, rectally, vagina administration, lung are administered and Usual any type of mucosa delivery.Additionally, be included within the scope of the invention, for mentioned in this article any one give The mode of medicine form is included in the invention.
Vaccine according to the present invention can be used once, or any number of times such as twice, three times, four times or five times.Use The vaccine once above effect with the immunne response strengthening gained.By by vaccine to be different from form or the body of previous administration Body region is administered, and vaccine can be reinforced further.Booster shot is the booster shot of homology or allos.Homology booster shot It is to comprise identical construct with ensuing vaccination, and more specifically comprise identical delivery vector, especially for the first time It is identical viral vector.Allos booster shot is to comprise identical construct in different viral vector.
Second active component
One aspect of the present invention is, vaccine combination provided herein and the second active component are used in combination.Vaccine group The administration of compound and the second active component can be in succession or united.Second is given above for both cancer and infection The example of active component.Also having the aspect to be, vaccine combination can specify relevant its of clinical disease to be treated He is used in combination in treatment.These treatments can include operation, chemotherapy or gene therapy, immunostimulation material or antibody；This area Technical staff can for specifying in the case of determine suitable therapeutic alliance.
In some cases, by the Therapeutic Method of the present invention and more medical therapy such as chemotherapy, radiotherapy, use The treatment of immunostimulation material, gene therapy, use antibody and/or the treatment of antibiotic and the therapeutic combination of use dendritic cell To be suitable.
Diagnosis and prognostic tool
Exploitation can widely used be provided by the peptide of the present invention about Cancerous disease and the diagnosis of infection and method of prognosis Basis.Therefore, in the embodiment that other are useful, the compositions of the present invention is in vitro or in-situ diagnostics table in individuality Reach the compositions of the existence of the cell of IDO.This diagnostic method is based on to IDO reaction-ive T cell in PBL or in tumor tissues Detection.
It thus provides a kind of IDO reactivity T in PBL or in tumor tissues in vitro or in-situ diagnostics individuality The diagnostic kit of the existence of cell, this test kit comprises one or more peptides of the present invention, and provides a kind of detection The method of the existence of this reaction-ive T cell in body, the method include the peptide of tumor tissues or blood sample and the present invention and I class or The complex contacts of the fragment of II class HLA molecule or this quasi-molecule and to detect described complex thin with described tissue or described blood The combination of born of the same parents.In one aspect, the invention provides the peptide of the present invention and I class or II class HLA molecule or the fragment of this quasi-molecule Complex, it can be used as all diagnostic reagents as described herein.Such complex can be monomer or polymer.
Another useful diagnosis or method of prognosis are based on generating antibody in heterogenous animal species, such as, for the present invention The murine antibody of people's IDO derived peptide, then it can be used to, and such as, the existence of the cancerous cell of this peptide is presented in diagnosis.For so Immune purpose, the amount of peptide can less than in vivo treatment during use amount, such as ones listed above amount.Generally, excellent The dosage of choosing can be about the peptide in 1 μ g-about 750 μ g range.It is likely to the peptide immunity based on using the present invention and produces monoclonal Antibody.Therefore, the invention also relates to the molecule of the peptide of the specific binding present invention, particularly monoclonal or Anti-TNF-α Body, including its fragment, and relates to block the molecule of such combination, such as, for the monoclonal or many of the peptide of the present invention Antibody produced by clonal antibody.The T that the invention also relates to the peptide of the specific binding present invention or the separation of protein is thin Born of the same parents' receptor and its code nucleic acid separated.Such φt cell receptor can such as use standard well-known to the ordinarily skilled artisan Technology is cloned from protein or peptide-specific T-cell.
In one aspect, the invention still further relates to comprise can specific binding IDO as herein described and/or its fragments of peptides The T cell of the separation of φt cell receptor.The T cell separated can be cd8 t cell or cd4 t cell.The T cell separated is preferably The T cell expanded the most in vitro.The method of amplification in vitro T cell is well-known to the ordinarily skilled artisan.Such T cell is permissible It is particularly used for shifting treatment cancer by adoptive transfer or autogenous cell.Therefore, the invention still further relates to the medicine comprising T cell Compositions and Therapeutic Method, the method includes comprise can the T of φt cell receptor of specific binding IDO or its fragments of peptides Cell is applied to the individuality needing its individuality such as to suffer from cancer and/or infection.Autogenous cell transfer can the most such as Walter etc., are carried out described in (1995).
Present invention provide for treating, prevent, alleviate or cure the clinical disease such as cancer being expressed as feature with IDO Disease and the mode of infection (preferably cancer), which includes using effective dose as defined herein to the individuality suffering from this disease Compositions, can the molecule of specific binding fragments of peptides (can antibody the most as herein described or φt cell receptor or many Component Kit).Therefore, another aspect of the present invention is to provide a kind for the treatment of and SEQ ID NO:1 and/or SEQ ID NO: The method expressing relevant clinical disease of the IDO of 16.
Monitoring immunization
In preferred embodiments, the pharmaceutical composition of the present invention is vaccine combination.The most interesting sum One aspect of the present invention is monitoring immunization in using the individuality of vaccine combination of the present invention.Therefore, medicine Compositions can be can to cause cancer and/or the immunogenic composition of the immunne response of infection or vaccine.Such as this paper institute Using, statement " immunogenic composition or vaccine " refers to cause for the cell such as cancerous cell, APC or DC expressing IDO The compositions of the immunne response of at least one type.Therefore, such immunne response can be following any one: CTL response, Wherein generation is capable of identify that the HLA/ peptide complexes presented on cell surface thus causes cytolytic CTL, i.e. vaccine Cause and the experimenter of inoculation produces the effector T cell to cancerous cell with cytotoxic effect；Anti-cancer antibody is caused to produce B cell response；And/or DTH type immunne response.It is an object of the invention to the compositions of the present invention be used by monitoring The immunity of described individuality is monitored in any of the above-described kind of reaction after individuality.
In one aspect, the method that the present invention relates to monitor immunization, said method comprising the steps of:
I) provide from individual blood sample,
Ii) provide IDO or its fragment, wherein said protein or peptide can be any one protein as herein described or Peptide,
Iii) determine whether described blood sample comprises the antibody being combined with described protein or peptide specific or containing with described The T cell of the φt cell receptor that protein or peptide specific combine, and
Iv) so that it is determined that created the immunne response for described protein or peptide in described individuality.
This individuality is preferably people, such as, with IDO or its fragments of peptides or code for said proteins or the nucleic acid immunization of peptide People.
Multicomponent kit (Kit of Parts)
The invention still further relates to a kind of multicomponent kit, it comprises
Any one vaccine combination as herein described, and/or
IDO albumen or its variant, and/or
Any one of the polypeptide fragment of IDO, its variant and/or peptide therefrom as described herein, and/or
Any one coding above-mentioned two bullets protein nucleic acid and about how using this multicomponent reagent The description of box.
The invention still further relates to a kind of multicomponent kit, it comprises
Any one vaccine combination as herein described, and/or
IDO albumen or its variant, and/or
Any one of the polypeptide fragment of IDO, its variant and/or peptide therefrom as described herein, and/or
The nucleic acid of the protein of any one coding above-mentioned two bullets and the second active component.
Preferably, the second active component is selected with being associated with clinical disease to be treated so that in the feelings for the treatment of cancer Under condition, the second active component selects in the most above-named chemotherapeutics.Similarly, if treatment microorganism/virus infects, Second active component is preferably antibiotic and/or antiviral agent.
The component of multicomponent kit is preferably included in independent compositions, but, owning of multicomponent kit Component is all contained in same compositions, and this is intended to be included within the scope of the present invention.Therefore the component of multicomponent kit can Or to be administered sequentially in any order simultaneously.
Detailed description
Fig. 1: such as the restricted t cell response of the HLA-A2-for IDO measured by IFN-γ ELISPOT.Analyze from 13 healthy individuals, 4 patient with breast cancers, 6 melanoma patients and the PBL of 10 renal cell carcinoma patients.All individualities are equal Positive for HLA-A2.Inspection peptide IDO2 (FLVSLLVEI) (a), IDO6 (VLSKGDGL) (b), and IDO5 (ALLEIASCL) (c). T lymphocyte peptide stimulates once, the most in duplicate with 4x105Cells/well is inoculated, and contains or not contain related peptides.Make With ImmunoSpot Series2.0 analyser (CTL analyser), every patient is calculated the average number of peptide specific speckle The number of IDO5 specific cell in the PBMC that (after deducting the speckle without adding peptide) (d) is measured by IFN-γ ELISPOT Express with the IDO in the PBMC measured by intracellular IDO dyeing and be associated.Patient is divided into two group: IDO-PBMC or IDO+ PBMC.Intracellular IDO expresses the single tail double sample t-inspection compareed by comparing MFIIDO and MFI homotype (MFIIsotype) and comes Being given, wherein MFI is average fluorescent strength.For p-value ＜ 0.05 (significance level), PBMC is defined as IDO+.White triangles Give IDO5-specific spots average/4x10 in each group5PBMC.Black triangles represents IDO5-specific spots in each group Average.E example that () is reacted from ELISPOT for IDO5 in the PBMC of patient with breast cancer.
The tetramer analysis of Fig. 2: IDO5 specific T-cells.(a), HLA-A2-restricted positive control peptide HIV- 1pol476-484(ILKEPVHGV) combination is compared by assembling mensuration with peptide IDO5.B (), from renal cell carcinoma patients The example of IDO5 specific C D8T cell in PBL, it uses tetramer complex HLA-A2/IDO5-PE and CD8-other algae indigo plant egg White dyeing by flow cytometry is imaged.As negative control, from the PBL tetramer complex HLA-of same patient A2/HIV pol476-484-PE and CD8-allophycocyanin dye.(c), after in vitro or in vitro peptide stimulates once, from Healthy donors or the PBL tetramer complex HLA-A2/IDO5 from breast carcinoma, melanoma cancer or renal cell carcinoma patients Or HLA-A2/HIV pol carries out dyeing and passing through flow cytometry.Dotted line explanation IDO5 tetramer sun in same patient Sexual cell all can detect that in the case of in vitro and external two kinds.(d), by flow cytometry image in vitro from nephrocyte CD45RA and CD28 of the IDO5 tetramer/CD8 gated cells (gated cell) in the PBMC rich in cd8 t cell of cancer patient The example of phenotype analytical.In order to compare, cell isotype matched control dyes.E (), the IL-2 from melanoma patients expands The example of TIL culture, it uses tetramer complex HLAA2/IDO5-PE and CD8-APC-Cy7 to pass through flow cytometry Dye and image.As negative control, TIL tetramer complex HLA-A2/HIV pol476-484-PE and CD8-APC- Cy7 dyes.F (), as the tetrameric positive control of IDO5, IDO5 specific T-cell clones HLA-A2/HIV-PE and HLA- A2/IDO5-PE tetramer staining.
The specificity of Fig. 3: IDO5-specific T-cell clones and Functional Capability.(RBS35) pass through51Cr-discharges algoscopy Measure.(a), unused peptide or the dissolving of the T2-cell with IDO5 peptide pulse.B (), is added without or adds HLA-I class specificity resisting The SL of the IDO+ of body W6/32, HLA-A2+ colon carcinoma cell line SW480, and IDO-, HLA-A2+ colon carcinoma cell line The dissolving of HCT-116.(c), be added without and add with IDO pulse or the IDO+, HLA-A2+ of cold T2-cell of non-pulse black The dissolving (inhibitor and target ratio=20: 1) (d) of melanoma cell line FM55M, from HLA-A2 positive AML patient enrichment The dissolving of AML-blast cell.Use CD19 respectively+And CD3+Microballon removes AML-blast cell, B cell from the bone marrow of AML patient And T cell.Use highly enriched AML-blast cell as adding or to be added without the target of HLA-I class specific antibody W6/32 thin Born of the same parents.All mensuration are carried out with E: T different ratios.E rectangular histogram that the intracellular IDO in () display cancerous cell line expresses.Data generation 3 tests of table.Intracellular IDO expresses by comparing MFIIDO (dark rectangular histogram) and MFI Isotype control (light rectangular histogram) Single tail double sample t-inspection is given, and wherein MFI is average fluorescent strength.Left: HCT-116 (p=0.300).Right: SW480 (p =0.002).
Fig. 4: show the rectangular histogram (dark rectangular histogram) that intracellular IDO dyes.Negative control combine with fluorescent dye two Anti-dyeing individually (light color rectangular histogram).Using Di to determine that IDO expresses, Di is defined as MFIPositive-MFIBackground/ 2xSDBackground, wherein MFI is average fluorescent strength.If Di ＞ 1, then cell is defined as the IDO positive21.(a), colon cancer Cell line HCT-116 (0,01), and SW480 (1,3) (b), breast cancer cell line CAMA-1 (1,3), and CAMA-1+IFN-γ (1,8), and (c), immature DC (0,2), and ripe DC (1,2).
Fig. 5: pass through51The IDO5-specific T-cell clones (RBS35) that Cr-release algoscopy measures kills IFN-γ and processes The Functional Capability of breast cancer cell line.HLA-A2 positive breast cancer cell lines CAMA-1 (a) and MDA-MB-231 (b) is at IFN- Dissolving before and after γ process.All mensuration are carried out with E: T different ratios.(c), left: to show before IFN-γ processes The rectangular histogram that in CAMA-1, intracellular IDO expresses afterwards.Data represent 3 tests.Intracellular IDO expresses by comparing Single tail double sample t-inspection of MFIIDO (dark rectangular histogram) and MFI Isotype control (light color rectangular histogram) is given, and wherein MFI is Average fluorescent strength.Top: CAMA-1 (p=0.020 and MFIIDO/MFI Isotype control=2.3).Bottom: CAMA-1+IFN- γ treats 25 (p=0.004 and MFIIDO/MFI Isotype control=3.5).Right: to show before and after IFN-γ processes The rectangular histogram that in CAMA-1, HLA-A2 expresses.Data represent 3 tests.HLA-A2 expresses by comparing MFIHLA-A2 (dark straight Side's figure) and single tail double sample t-of MFI Isotype control (light color rectangular histogram) check and be given.Top: CAMA-1 (p=0.004 and MFIHLA-A2/MFI Isotype control=43.7).Bottom: CAMA-1+IFN-γ process (p=0.002 and MFIIDO/MFIHLA- A2=141.2).D (), by IDO5-specific T-cells bulk cultures, uses the IDOShRNA for lowering IDO protein expression The dissolving of the colon carcinoma cell line SW480 of transfection.As positive control, it is used as target with the SW480 cell of comparison ShRNA transfection thin Born of the same parents.All mensuration are carried out with E: T different ratios.(d), display comparison ShRNA (p=0.001 and MFIIDO/MFI Isotype control =4.8) SW480 that (top) and IDO ShRNA (p=0.040 and MFIIDO/MFI Isotype control=2.1) (bottom) transfect In intracellular IDO express rectangular histogram.
Fig. 6: pass through51The IDO5-specific T-cell clones (RBS35) that Cr-release algoscopy measures kills the function energy of DC Power.(a), autologous immaturity and the dissolving of ripe DC.(b), the allochthonous immaturity of HLA-A2+ and the dissolving of ripe DC.Institute Mensuration is had to carry out with E: T different ratios.(c), the rectangular histogram that in display DC, intracellular IDO expresses.Data represent 3 tests.Carefully Intracellular IDO is expressed by comparing MFIIDO (dark rectangular histogram) and single tail double sample t-of MFI Isotype control (light color rectangular histogram) Inspection is given, and wherein MFI is average fluorescent strength.Left: external immature DC (p=0.100).Right: maturation in vitro DC (p= 0.001).D (), from autologous CD14+ mononuclear cell, CD3+T cell and the CD19+B cell of the direct isolated ex vivo of IDO+PBMC Dissolve.As comparison, we use autologous IDO-immature DC and IDO+ maturation DC of external generation.E (), as passed through In the PBMC from patient with breast cancer that ELISPOT measures, the HLA-A2 for EBV BMLF1280-288 (GLCTLVAML) limits The example of property t cell response processed.The culture of PBMC IFN-γ processes 5 days, does not contains (left) and containing 26IDO-specificity T Cell (PBMC: the IDO-specific T-cells ratios with 3000: 1) (right), then checks for the HLA-A2 from EBV BMLF1 The reactivity of restricted epitope (GLCTLVAML).Check three different PMBC concentration: 1.5x105Cell, 5x104Cell (on Face two row) and 104Cell (following two row).
Fig. 7: pass through51The specificity of the IDO5-specific T-cells that Cr-release algoscopy measures and Functional Capability: (a), RBS35 is to being added without and adding with IDO peptide or the HLA-A2 of the cold T2-cell of uncorrelated peptide (HIV-1pol476-484) pulse The dissolving (inhibitor and target ratio=20: 1) of +/IDO+ K-1735 FM55M, and use natural killer cell system The NK cytoactive of the RBS35 that K562 measures as target cell.B (), the RBS35 AML-to being enriched with from HLA-A2+AML patient is female The dissolving of cell.Use CD19 respectively+And CD3+It is thin that microballon removes AML-blast cell, B cell and T from the bone marrow of AML patient Born of the same parents.Use highly enriched AML-blast cell as the target cell adding or being added without HLA-I class specific antibody W6/32. (c), with IDO peptide or the dissolving of the T2-cell of uncorrelated peptide (HIV-1pol476-484) pulse, and by IDO5-specificity T- The dissolving to HLAA2+/IDO+ colon carcinoma cell line SW480 of the cell bulk cultures.D (), passes through51Cr-release algoscopy measures By three kinds of different IDO5-specific T-cell clone (RBS26 (white triangles), RBS31 (black triangles), RBS46 (ash Color triangle)) molten to HLA-A2+/IDO+ colon carcinoma cell line SW480 and HLA-A2+/IDO-colon carcinoma cell line HCT-116 Solve.All mensuration are carried out with E: T different ratios.
Fig. 8: by the Clustal W multiple ratio pair to IDO sequence
Fig. 9: by the Clustal W pairing comparison to IDO and IDOLIKE
Embodiment
Embodiment 1
Patient/individuality
PBL/PBMC gathers from cancer patient's (breast carcinoma, melanoma and renal cell carcinoma) and normal healthy controls.At any sort Type anticancer therapy extracts blood sample after terminating minimum 4 weeks.Most of renal cell carcinoma patients had used IL2 and IFN-α treatment in the past, greatly Most melanoma patients have accepted IL2 and the IFN-α of too high dose, and the chemotherapy of all patient with breast cancer's several types (such as, epirubicin, Docetaxel, capecitabine), Herceptin and/or incretotherapy are treated in advance.Use and drench Bar cell separation agent (Lymphoprep) partition method separates PBL, HLA typing (Department of Clinical Immunology, University Hospital, Copenhagen, Denmark) and it is chilled in the FCS containing 10%DMSO In.Including amounting to 20 HLA-A2+ patients, before blood sampling, they all do not accept immunization therapy.Carrying out these remedy measures Any one measure before obtain informed consent from these patients.
Peptide
According to about HLA-A2 allelic preferred peptide length, anchor residues and auxiliary anchor residues (auxiliary Anchor) knowledge prediction is from the epi-position of IDO.Use " data base SYFPEITHIP "32MHC I class grappling is checked in conjunction with manual The protein sequence of residue, carries out the scanning of IDO albumen.The peptide selected is purchased from Genscript.Produce 11 synthesis 9mer and 10mer peptide: IDO1 position 54-62 (QLRERVEKL), IDO2 position 164-172 (FLVSLLVEI), IDO3 position 195-203 (TLLKALLEI), IDO4 position 41-49 (FIAKHLPDL), IDO5 position 199-207 (ALLEIASCL), IDO6 position 320- 328 (VLSKGDGL), IDO7 position 383-391 (DLMNFLKTV), IDO8 position 275-283 (VLLGIQQTA), IDO9 position 101-109 (KVLPRNIAV), IDO10 position 61-70 (KLNMLSIDHL) and IDO11 position 341-350 (SLRSYHLQIV). Peptide is dissolved in DMSO (final concentration 10mM) or in distilled water (final concentration 2mM).HLA-A2 high-affinity is used to combine epi-position HIV-1pol476-484 (ILKEPVHGV) is as uncorrelated comparison.HLA-A2 restricted Epstein-Barr virus peptide EBVBMLF1280-288 (GLCTLVAML) with comparing.
The assembling algoscopy of the peptide for being combined with MHC I quasi-molecule
As previously mentioned33In assembling algoscopy, measure synthetic peptide (Genscript) and use [35S]-Methionine metabolism mark The binding affinity of the HLA-A2 molecule of note.This algoscopy is based on the sky discharged from TAP deficient cells system T2 after cell dissolves Peptide-mediated the stablizing of HLA molecule.The stable HLA molecule folded is by using HLA I class specific conformation dependency monoclonal Antibody (mAb) W6/32 immunoprecipitation is also separated by isoelectric focusing (IEF) gel electrophoresis.Use ImageGauge Phosphoimager program (FUJI Photo Film, Carrolton, TX) quantitatively MHC (MHC) Heavy chain band.The intensity of band is directly related with the amount of the I class MHC complex that the peptide reclaimed during measuring is combined.HLA-A2 Recovery measure in the presence of 100,10,1 and 0.1 μM of related peptides.For every kind of peptide, C50 value is calculated as sufficiently achieving half The peptide concentration of number maximum stable.
The antigenic stimulus of PBL
In order to expand the sensitivity that ELISpot (ELISPOT) measures, PBL stimulates 1 with peptide in vitro before analysis Secondary34.At the 0th day, PBL is thawed, and with 2x106The concentration of cell in 24 orifice plates (Nunc) with 2ml/ hole at 10 μMs of peptides (GenScript) it is seeded in the presence of in the X-vivo culture medium (BioWhittaker) containing 5% heat inactivation human serum.1 After it, in culture, add 40IU/ml recombinant interleukin-2 (IL-2) (PeproTech).Cultivate cell the 8th day IFN-γ ELISPOT tests in measuring.
IFN-γ ELISPOT algoscopy
As mentioned previously17, ELISPOT algoscopy is used for the effector lymphocyte of quantitative release peptide epi-position-specificity INF-γ.? In some tests, as described(34)PBMC peptide stimulates 1 time in vitro to expand the sensitivity measured before analysis.Letter Yan Zhi, 96 orifice plates (the MultiScreen MAIP N45 of celluloid shop fixtures；Millipore) it is coated with anti-IFN-γ Ab (1-D1K；Mabtech).Each hole is washed, is closed by X-vivo culture medium, add in duplicate with different cell concentrations Enter effector lymphocyte, add or be added without 10 μMs of peptides.Each plate is incubated overnight.Second day, discard culture medium, wash each hole, then add Enter the anti-(7-B6-1-Biotin of biotinylation two；Mabtech).Each plate is incubation 2h under room temperature (RT), washing, and in each hole Add Avidin-enzyme conjugates (AP-Avidin；Calbiochem/Invitrogen Life Technologies).Each plate exists Incubation 1h under RT, and in each hole, add zymolyte NBT/BCIP (Invitrogen Life Technologies) and at RT Lower incubation 5-10min.After darkviolet speckle occurs, by terminating reaction with tap water washing.Use ImmunoSpot Speckle is counted and can calculate peptide from the number of the cell forming speckle by Series2.0 analyser (CTL analyser) Specific CTL frequency.
Flow cytometry
For tetramer staining, from cancer patient and the PBL of healthy donors and from cancer patient TIL in vitro Stimulate 1 time with peptide, or the most in vitro analysis.Dynal CD8 feminine gender separating kit (Dynal Biotech) is used at the 7th day Cd8 cell is separated from PBL.The tetramer staining that the T cell culture PE obtained combines, then with fluorescent dye combination MAb carries out antibody staining: CD8-allophycocyanin/APC-Cy7, CD3-FITC, CD3-FITC, CD45RO-FITC, CD45RA- PE-Cy5 and CD28-allophycocyanin (BD Immunocytometry Systems).Tetramer staining in PBS+2%FCS, RT, lucifuge carries out 15min, and antibody staining is in PBS+2%FCS, and 4 DEG C, lucifuge is carried out.The MHC tetramer complex used For: HLA-A2/IDO5 (ALLEIASCL) and HLA-A2/HIV-1po1476-484 (ILKEPVHGV).Sample is at BD FACS Aria upper use DIVA software (BD Biosciences) analyzes.
The IDO of cancerous cell line and DC expresses to use flow cytometry to check.At fixing and saturatingization (Cytofix/ Cytoperm, BD) after, cell dyes with little mouse-anti IDO antibody (Millipore Corporation), then uses FITC-labelling Anti-mouse two anti-(DAKO) dyeing.For all tests, including the two stain-fast negative controls only combined with FITC-, with Determine and autofluorescence reasons for its use fluorescence anti-by two adhered to mistakenly.Di is used to determine that IDO expresses, this dyeing Index definition is MFIPositive-MFIBackground/2xSD Background, wherein MFI is average fluorescent strength.If Di ＞ I, then cell is fixed Justice is that IDO is positive21。
The HLA-A2 of cancerous cell expresses to use flow cytometry to check.HLA-A2mAb (the BD that cell combines with fluorescent dye Bioscience) dyeing.For comparing, cell isotype matched control dyes.Sample uses DIVA soft on BD FACS aria Part (BD Biosciences) is analyzed.Assuming normal state, HLA-A2 expresses by the list comparing MFIHLA-A2 and MFI Isotype control The inspection of tail double sample t-is given, and wherein MFI is mean fluorecence 9 intensity.For p-value ＜ 0.05 (significant level), cell defines For HLA-A2+.The multiple (the fold of expression) expressed is defined as MFIHLA-A2/MFI Isotype control.
Dendritic cell (DC)
By adhering to 60min on culture dish at 37 DEG C in the RPMI-1640 rich in 10% people's AB serum from PBMC Produce DC.Adhere to mononuclear cell in the RPMI-1640 culture medium that with the addition of 10% people's AB serum at IL-4 (1000U/ml) Cultivate 6 days with in the presence of GM-CSF (800U/ml).By adding IL-1 β (2ng/ml), IL-6 (1000U/ml), TNF-α (10ng/ml) make DC ripe with PGE2 (1 μ g/ml).
T cells with antigenic specificity culture and the foundation of clone
PBL from cancer patient uses (25Gy), autologous DC (PBL: the DC ratio=3x10 of IDO5-load irradiated6 ∶3x105) and 3 μ g/ml β 2m, 20U/ml IL-12 (PeproTech) and 40U/ml IL-7 (PeproTech) stimulate.Culture Within every 10 days, stimulate with the autologous DC (2 ×) irradiated, then stimulate with the PBL (2 ×) irradiated.Add after stimulating with DC every time 20U/ml IL-12 (PeproTech) and 40U/ml IL-7 (PeproTech), and after stimulating, add 40U/ with PBL every time ml IL-2(PeproTech).After January, in standard51Cr-release detects the grown culture specificity to IDO5 in measuring.Come From the PBL of specific culture 106PBL and the 120U/ml IL-2's (PeproTech) of (25Gy) IDO5 load that/ml irradiates In the presence of cloned by limiting dilution.Every 3-4 days, add 50 μ l fresh cultures, make containing IL-2 final concentration of 120U/ml.Use the PBL (5x10 of IDO5 load4Cells/well) and the clone of 120U/ml IL-2 amplification growth.After amplification, gram Grand in standard51Cr-release detects specificity and cytotoxic potentials in measuring.
Cytotoxic assay
As described in elsewhere35Carry out the Cytotoxic routine for CTL mediation51Cr-discharges mensuration.Target cell is that T2-is thin Born of the same parents, the autologous immaturity of external generation and ripe DC, allogeneic HLA-A2 positive immaturity and ripe DC, autologous in vitro dividing From mononuclear cell, T cell and B cell (use CD14+, CD3+ or CD19+ microballon (MACS) to separate), natural killer cell Target cell system K562, the HLA-A2 positive AML-blast cell of in vitro enrichment (uses CD19+ and CD3+ microballon (MACS) to be isolatable from The bone marrow of AML patient), HLA-A2 positive breast cancer cell lines CAMA-1 and MDA-MB-231, HLA-A2 positive colon cancer cell It is that HCT-116 and SW480 (all can be at American Type Culture Collection (American Type Culture Collection) (ATCC) obtains) and HLA-A2 positive K-1735 FM55M (from IPD-ESTDAB data base, Obtain at www.ebi.ac.uk/cgi-bin/ipd/estdab/36).Use HLA specificity mAb W6/32 (2 μ g/100 μ l) resistance Disconnected dissolving37.In some measure, cancerous cell 100U/ml IFN-γ processes 2 days.
The enrichment of AML blast cell
We use CD19+ and CD3+ microballon (MACS) to remove B cell and T cell from the bone marrow of AML patient respectively.? Standard51Cr release uses highly enriched AML-blast cell (CD3-, CD19-) as target cell in measuring.
The downward of IDO in cancerous cell
People SW480 uses FuGene6 (Roche) with the shown short hairpin RNA available from SuperArray (ShRNA) plasmid Description transfection according to production firm.Cell directly dissolves in LSB buffer (Sigma).LSB lysate boils 5min And it is supported on 10% prefabricated protein gel (BioRad).Protein passes through half-dried transfer method (semidry transfer Method) electrotransfer is to pvdf membrane (Millipore Corporation) and with shown antibody saying according to production firm Bright book detects.Trace uses the ECL system available from Amersham and CCD camera (LAS-1000, Fnjifilm) development. Use following antibody: anti-Cdk7 (MO-1) (Santa Cruz) and anti-IDO (Millipore Corporation).
The HLA-A2-restricted T-cell epitope that IDO-is derivative
Use 11 IDO-derived peptide of algorithms selection based on main HLA-A2 specificity anchor residues and close subsequently Become16.Using ELISPOT IFN-γ secretion to measure, then we examine the peripheral blood T from cancer patient and healthy individuals The existence of the specific T-cells response for these IDO-derived peptide of cell.This mode had turned out effectively in the past For identifying tumor-specific cytotoxicity T-lymphocyte (CTL) in cancer patient17-19.Therefore, positive from HLA-A2, late The peripheral blood lymphocyte (PBL) of phase cancer patient (breast carcinoma, melanoma and renal cell carcinoma) is detecting it by ELISPOT Before stimulate 1 time with different peptide in vitro.As described17,20, select this step to expand the sensitivity of ELISPOT.Detect for IDO2(IDO164-172；FLVSLLVEI)、IDO6(IDO320-328；And especially IDO5 (IDO199-207 VLSKGDGL)； ALLEIASCL) ELISPOT reaction (Fig. 1).As comparison, we examine the PBL from healthy individuals for these three The reactivity of IDO derived peptide.In any one normal healthy controls, it is not detected by the spontaneous reaction for any one IDO derived peptide. Use " ncbi database " that the aminoacid sequence of these peptides is carried out blast search, show that these motifs are in IDO albumen only Dominant.
Embodiment 2
(material and method are as described in example 1 above)
The restricted T cell of IDO-reactivity HLA-A2-is detected cancer patient
Measured by assembling, by with the HLA-A2 high-affinity i.e. HIV-1pol476-484 of positive control epi-position (ILKEPVHGV) compare, check immunogenicity the most obvious IDO-derived peptide i.e., the affinity of IDO5 Yu HLA-A2 (Fig. 2 a).It should be noted that the HLA-A2 that IDO5 combines is even more preferable than high-affinity comparison epi-position.IDO5's Yu HLA-A2 High binding affinity enables us to prepare the stable HLA-A2/IDO5 tetramer, and it is used for passing through Flow cytometry IDO-reactivity CTL.This analysis clearly confirms that IDO5-reactivity cd8 t cell in the blood of HLA-A2 positive cancer patients Existence (Fig. 2).Fig. 2 b illustrates and is using by the HIV tetramer-composite body compared in renal cell carcinoma patients The example of IDO5 specific T-cells response after external stimulus.At the frequency being significantly increased IDO-reaction-ive T cell by stimulated in vitro While rate, in the patient selected IDO-reaction-ive T cell can easily Testing in vitro to (Fig. 2 c): in vitro after stimulation Having in three patients of the strongest response, respective reactivity also detects in vitro.In a word, analyze from 7 HLA- The individual PBL with 11 HLA-A2 positive patients of A2 positive healthy, shows that after stimulated in vitro, whole CD8+T are thin in cancer patient The average frequency of the IDO reactivity cell of born of the same parents is 0.03%, and by contrast, healthy donors is 0.001% (Fig. 2 c).
IDO-reaction-ive T cell (Fig. 2 b) all it is not detected by arbitrary healthy donors.IDO-reaction-ive T cell from Body dyeing shows that naturally occurring IDO5-specific T-cells has CD45RA-CD28+ maincenter/effector memory phenotype(34)。 The example of this in vitro phenotype dyeing of IDO tetramer gated cells shows in figure 2d.As a comparison, sample homotype coupling is right According to dyeing.Next step, we are examined from HLA-A2+ melanoma and incidence cancer patient by tetramer staining The existence of IDO-specific T-cells in the TIL culture that IL-2 processes.As illustrated in Fig. 2 e, IDO5-specific T-cells can To be easily detected in TIL.In a word, 4 in 5 patients analyzed can detect that IDO5-specific T-cells.With Sample ground, can detect from the IDO5-in the TIL culture of melanoma and incidence cancer patient special in ELISPOT Specific T cell (data do not show).In order to control the tetrameric specificity of HLA-A2/IDO5, we have dyeed IDO5-specificity T-cell clone.The HLA-A2/IDO5 tetramer dyes IDO5-specific T-cell clone the most effectively, and T-cell clone Not by comparison HLAA2/HIV tetramer staining (Fig. 2 f).
Embodiment 3
(material and method are as described in example 1 above)
The Functional Capability of IDO specific T-cells
After identifying patient's host response for IDO peptide, we use the PBL from these patients to produce pin in vitro CTL bulk cultures thing to this peptide.PBL is stimulated by the DC of autologous IDO5-pulse.After taking turns stimulation 4, discharge in standard 51Cr Mensuration detects peptide specific.Thin from the TAP-deficiency T2-of the cell dissolving IDO peptide pulse of these bulk cultures things Born of the same parents.In order to analyze the solvability of IDO specific T-cells in more detail, by limited dilution cloning method from these bulk cultures Thing sets up ctl clone.At of short duration amplification after date, in the release of standard 51Cr measures, analyze the specificity of the clone of growth.? In 33 T cell clones of display IDO SL ability, 4 clones are selected to be used for amplification further, because they have Preferably growth rate.Representational T cell clone draw in fig. 3 a: T-cell clone RBS35 effectively kills IDO5-arteries and veins The T2-cell of punching, and the T2-cell without peptide is not dissolved (Fig. 3 a).
Embodiment 4
(material and method are as described in example 1 above)
IDO-specific T-cells kills tumor targets
Dyeed by intracellular protein and then expressed by the IDO of the many cancerous cell lines of facs analysis inspection and DC21.For This, colon carcinoma cell line SW480, K-1735 FM55, breast cancer cell line CAMA-1 and MDA-MB231, directly richness It is positive that the AML-blast cell of collection and ripe DC are IDO.Only colon carcinoma cell line HCT-116 and immature DC are that IDO is negative.This Outward, the IFN-γ of cancerous cell line processes increases IDO expression.
In the representative example diagram of IDO dyeing rectangular histogram in the diagram.
It is essential that T cell clone RBS35 not only kills the T2-cell of peptide pulse with high effect, but also kill HLA- A2+, IDO+ colon carcinoma cell line SW480 (Fig. 3 b).By contrast, RBS35 does not dissolves HLA-A2+/IDO-colon carcinoma cell line HCT-116 (Fig. 3 b).By using HLA specificity mAbW6/32 to block HLA-I class, completely eliminate SW480 target cell Dissolve it was confirmed the HLA-of RBS35 restricted (Fig. 3 b).Similarly, RBS35 kills HLA-A2+/IDO+ K-1735 FM55M (Fig. 3 c).The cold targeted inhibition using the unmarked T2-cell of IDO (10 μMs) peptide pulse measures and confirms killing effect HLA-A2/ peptide specific: the T2-cell adding cold (unmarked) IDO5-pulse completely eliminates FM55M melanoma Killing of cell, and add the cold T2-cell without peptide and killing of FM55M is not affected (Fig. 3 c).Add and use uncorrelated peptide (HIV-1pol476-484) killing of FM55M is not the most affected (Fig. 7 a) by the cold T2-cell of pulse.Do not observe for The cytotoxicity (Fig. 7 a) of NK-cellular targets cell line k562.
Additionally, we have detected RBS35 dissolves the people HLA-A2+AML-mother that the bone marrow from AML patient is enriched with the most in vitro The ability of cell.For this purpose it is proposed, we remove T cell (CD3+) and B cell (CD19 from the bone marrow of HLA-A2+AML patient +)；The most highly enriched AML-blast cell (CD3-, CD19-) is used as51Target cell in Cr release mensuration.Such as institute in Fig. 3 d Showing, RBS35 dissolves leukaemia effectively in the dependent mode of HLA.We are from 6 patients (5 HLA-A2+ patients and 1 Name HLA-A2-patient) in be enriched AML blast cell, and all these cell expresses IDO (data do not show).RBS35 with The dependent mode of HLA dissolves HLA-A2+ leukaemia effectively, and HLA-A2-leukaemia is not dissolved (Fig. 7 b).
In order to illustrate that RBS35 causes the representativeness of tumor targets to be killed, polyclone IDO5-specificity bulk cultures thing and Three kinds other T cell clone (RBS26, RBS31, the RBS46) SW480 that causes kill display in Fig. 7 c and Fig. 7 d.Class Being similar to RBS35, any one in these clones (RBS26, RBS31, RBS46) does not all dissolve HLA-A2+/IDO-colon cancer cell It is HCT-116 (Fig. 7 d).
Finally, we examine killing of HLA-A2+ breast cancer cell line CAMA-1 and MDA-MB-231.CAMA-1 cell System is killed (Fig. 5 a) by RBS35, and MDA-MB-231 is not identified (Fig. 5 b) by RBS35.INF-γ processes increases by two kinds of cell lines In IDO express.Consistent with this, INF-γ processes increases killing of the CAMA-1 that causes of RBS35, and introduces MDA- The killing effect (Fig. 5) of MB-231 cell.
It addition, we show that polyclone IDO5-specificity bulk cultures thing and RBS35 clone the killing effect caused really It is that IDO-is specific.Therefore, using IDO shRNA, we make under the IDO protein expression in people's SW480 colon carcinoma cell line Adjust and thus saved these tumor cells in order to avoid be killed (Fig. 5 c).This downward is dyeed by intracellular protein and is manifested. These dyeing confirm that the use of IDO ShRNA reduces the level (Fig. 5 d) of IDO protein expression in cell.Subsequently, transfection is thin Born of the same parents are used as51Target cell in Cr-release mensuration.Cancerous cell with IDO ShRNA transfection is not large quantities of by polyclone IDO-specificity Culture identification, and be killed, as shown in Figure 5 c with the cell of uncorrelated comparison ShRNA transfection.
Embodiment 5
(material and method are as described in example 1 above)
Immunologically competent cell is killed by IDO-specific T-cells
IDO expresses and is not limited to tumor and stromal cells, and can also be induced in immunocyte.Accordingly, as Next and the most even more important step, whether the DC that we manage to solve to express IDO is also easy to be killed by IDO-reactivity CTL Dead problem.In order to test this viewpoint, we create autologous DC from the identical donor having produced ctl clone；By adding The standard Maturation mixture being made up of IL-1 β, IL-6, TNF-α and PGE222 makes DC ripe22.RBS35 effectively kills maturation DC.Comparing, autologous immaturity IDO-DC is not killed (Fig. 6 a) by RBS35.And, we examine RBS35 to from HLA-A2+ IDO+ maturation DC of donor and the identification of IDO-immature DC.The DC of allogeneic maturation is killed by RBS35, and from identical The IDO-immature DC of donor is not killed (Fig. 6 b).
In fig. 6 c, it is illustrated that mDC expresses IDO, contrary with iDC.Next step, we have detected RBS35 and dissolve autologous list The ability of nucleus, T cell and B cell.For this purpose it is proposed, we from IDO+PBMC direct isolated ex vivo CD14+ mononuclear cell, CD3+T cell and CD19+B cell.The cell separated is subsequently used as the target cell during 51Cr-release measures.Autologous CD14+ monokaryon Cell, CD3+T cell and CD19+B cell are not dissolved (Fig. 6 d) by RBS35.
Finally, we establish an external model to check whether that IDO-specific T-cells expresses IDO's by removing SC strengthens immunne response.Therefore, the culture IFN-γ of PBMC processes to increase containing and does not contains autologous Immunocompetence and IDO in the culture of IDO specific T-cells express.After 5 days, we examine in culture for coming Immunoreactivity from the restricted immunodominant epitope of HLA-A2 of EBV BMLF1280-288 (GLCTLVAML).Although in training It is identical for supporting total cell number in thing, but reactivity for EBV peptide is higher in the culture containing IDO-specific T-cells (Fig. 6 e).Next step, whether we have gone through the addition of IDO specific T-cells increases to immunoreactivity permission and exists ELISPOT only uses 10 far below normal detection limit4EBV is reacted the degree detected by PBMC.It is true that we can Even to detect under the PBMC of this low concentration that obvious EBV reacts (Fig. 6 e).The most like that, we are low cell at this In the culture without IDO-specific T-cells, can't detect any EBV under concentration react (Fig. 6 e).
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Claims (8)

1. a vaccine combination, it comprises:
A) 18-25 the continuous amino acid of SEQ ID NO:1, the immunogenic activity fragments of peptides that comprises SEQ ID NO:6；With
B) adjuvant,
It is used as medicine.
Vaccine combination the most according to claim 1, wherein said adjuvant choosing freely adjuvant based on DNA of bacteria, based on The adjuvant of oil/surfactant, based on the virus adjuvant of dsRNA, imidazoquinolie and the group of GM-CSF composition.
Vaccine combination the most according to claim 2, wherein said adjuvant is Montanide ISA adjuvant.
Vaccine combination the most according to claim 3, wherein said adjuvant is Montanide ISA 51 or Montanide ISA 720。
5. the peptide separated, it is 18-25 the continuous amino acid of SEQ ID NO:1, and it comprises SEQ ID NO:6's Sequence.
6. effective dose according to the vaccine combination described in any one of claim 1-4 or separation according to claim 5 Peptide manufacture for treatment or prophylaxis of cancer medicine in purposes.
Purposes the most according to claim 6, the peptide of wherein said vaccine combination or separation is prepared for and other cancer Disease treatment associating.
Purposes the most according to claim 7, the most other treatment of cancer choosing free chemotherapy, X-ray therapy, use The group of the treatment composition of the treatment of immunostimulation material, gene therapy, the treatment of use antibody and use dendritic cell.
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